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REVIEW OF
Neurobiology
Volume 101
SERIES EDITORS
R. ADRON HARRIS
Waggoner Center for Alcohol and Drug Addiction Research
The University of Texas at Austin
Austin, Texas, USA
PETER JENNER
Division of Pharmacology and Therapeutics
GKT School of Biomedical Sciences
King’s College, London, UK
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ISBN: 978-0-12-387718-5
ISSN: 0074-7742
Numbers in parentheses indicate the pages on which the authors’ contributions begin.
xi
xii CONTRIBUTORS
This volume of the International Review of Neurobiology describes the state of the
art and the future of biomarkers in neurological and psychiatric diseases. Cur-
rently, the diagnosis for all neuropsychiatric disorders is carried out by psychia-
trists via interview, observation, and classification of patients who typically have
heterogeneous symptoms and medical histories. The recent emergence of molec-
ular and image-based biomarkers for these conditions would therefore greatly
facilitate disease diagnosis and stratification. This may require deconstruction of
the existing long-standing procedures aimed at classification of broad patient
categories in favor of identifying biomarker-defined disease subtypes. Ultimately,
this will assist in personalized medicine approaches and may be facilitated by
developments in the areas of biosensors, neuroinformatics, and e-neuropsychiatry.
The first chapter by Filiou and Turck introduces the content of the volume by
describing how biomarkers are now in demand in neuropsychiatric research for
diagnosis, treatment response monitoring, and development of novel therapeutics.
However, biomarker discovery in this field is challenging due to the fact that these
are complex disorders, information on the affected molecular pathways is scarce,
and there is considerable interpatient heterogeneity within a given disorder and
overlap of symptoms across different conditions. Because of this disease complexi-
ty, a panel of biomarkers derived from multiple platforms will be needed to define
these conditions at the molecular level. Ultimately, the coordinated effort of
researchers, physicians, funding organizations, and standardization initiatives
will be needed to overcome these challenges.
The second chapter by Owen and Matthews describes recent advances
in imaging technologies to study neuroinflammatory, neurodegenerative, and
neuropsychiatric conditions such as multiple sclerosis, Alzheimer’s disease,
Parkinson’s disease, stroke, and schizophrenia. The main technique described,
positron emission tomography (PET), is used to study the proliferation of
microglia in these conditions as this is a stereotyped response after a variety of
pathological insults. There has been significant interest in quantifying microglial
density in vivo in research and clinical decision making. However, this has
been hindered by the lack of appropriate radioligands. With recent development
of several new generation ligands with improved specific binding, this now
xv
xvi PREFACE
seems possible and should enable PET to become a more valuable tool for use
in the clinical studies of such neuropsychiatric disorders.
The third chapter by Woelk et al. covers the use of gene expression analysis of
blood cells for diagnosis of neuropsychiatric disorders. The authors carry out a
review of studies which have analyzed gene expression in blood cells from patients
with neuropsychiatric disorders with an emphasis on developing diagnostics for
schizophrenia. The authors also discuss the future directions of the field including
using microRNA expression for developing diagnostic classifiers and the potential
use of blood cell gene expression patterns to tailor antipsychotic medications to
individual patients. They also describe the likely future impact of next-generation
sequencing technologies. As the costs diminish and software tools for analysis of
this high-content data becomes more available, the possibility of developing more
accurate classification tools for neuropsychiatric disorders will increase.
The fourth chapter by Martins-de-Souza et al. describes the state of the art and
possible future developments in the use of proteomic technologies for the study of
neuropsychiatric conditions and for the development of novel molecular diagnostic/
prognostic tests. Such advances have already been partly achieved for illnesses such
as cancer although they have had a less profound impact in the case of neuropsy-
chiatric disorders such as schizophrenia. The authors discuss the pressing need for
more sensitive and accurate technologies with overall importance on technologies
which can be used for validation and implementation of the resulting biomarkers as
simple and effective tests for use in the clinical environment. Such advances will put
proteomics closer to clinical applications in the neuropsychiatry field.
The fifth chapter by Chan et al. gives an update on emerging evidence
for identification of blood-based molecular biomarkers in schizophrenia. The
authors have combined a review of the literature with the results of a comprehen-
sive in-house study showing the identification of candidate blood-based biomar-
kers for schizophrenia and for antipsychotic drug response. Taken together, the
findings suggest that there are effects on the immune system and inflammation
response in schizophrenia. The findings also suggested that there is an activation
of the stress response, as shown by increased levels of cortisol and activation of the
hypothalamic–pituitary–adrenal (HPA) axis in patients. It is expected that such
biomarkers will prove useful as an additional means of characterizing specific
immune, metabolic, or hormonal pathways in schizophrenia, which should
pave the way for development of future patient stratification and personalized
medicine strategies.
The sixth chapter by Guest et al. describes the finding of abnormalities in
metabolism and hormonal function in patients with schizophrenia. The authors
describe decades of research converging on the fact that the pathogenesis of
schizophrenia can involve perturbations in metabolic and HPA axis pathways in
some patients. The observed differences in manifestation of these effects could be
related to differences in symptoms between patients and in responses to
PREFACE xvii
studies. Patient-derived induced pluripotent stem cells and adult stem cells from
the olfactory tissue in the nose have already been used to provide novel insights
into a number of brain diseases. This work was originally inspired by the obser-
vation that the sense of smell is impaired in many brain diseases, including
neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease,
and neuropsychiatric disorders, such as schizophrenia. These findings suggest that
biomarker discovery may be possible from investigating such disease-associated
cells in the form of patient-derived stem cells. Such cellular models carry the
disease phenotypes and span the variability encountered across patient popula-
tions. They also provide potential experimental tools to identify novel molecular
biomarkers that distinguish patients from controls and may therefore lead to
development of empirical tests for differential diagnosis or for monitoring disease
progression.
The 10th chapter by Schwarz et al. describes the recent advances in applying
multiplexed immunoassay systems to identify molecular diagnostics for psychiat-
ric disorders. The authors describe approaches to identify disease-related molec-
ular abnormalities when there is uncertainty regarding the validity of the clinical
diagnosis. They also present an introduction to the multiplex immunoassay
approach that facilitates identification and quantitation of molecular biomarkers
and also for extending these molecular findings into the realms of identifying the
associated functional consequences. As chronic sufferers of neuropsychiatic dis-
eases are likely to have a poor prognosis, an accurate molecular test may lead to
early intervention and thereby improve patient outcomes. A molecular test would
also open up the possibility of stratifying patients more accurately which is crucial
for personalized medicine approaches.
The 11th chapter by Izmailov et al. gives a description of algorithm develop-
ment for diagnostic biomarker assays. As a test case, they present the ground-
breaking development of a serum-based test to help confirm the diagnosis of
schizophrenia. They identified a multiplex panel of 51 immunoassays which
allowed reproducible identification of schizophrenia patients compared to con-
trols with high performance. Validation of this test involved development of a
linear support vector machine decision rule and they tested the performance of
this using cross-validation. This resulted in readjustment of the panel and algo-
rithm to a smaller set of assays, and they developed a simple procedure for
maintenance and recalibration of the assays across time. The resulting decision
rule delivered a sensitive and specific test for presence of schizophrenia compared
to controls. The next stage will be to carry out large-scale clinical validation
studies using samples from more diverse psychiatric patient populations and
settings in a series of prospective studies for translation to the clinical setting.
The 12th chapter by Bahn et al. describes the challenges of introducing new
biomarker products for neuropsychiatric disorders into the market place. The
general opinion is that improvements over the current subjective tests are
PREFACE xix
essential. Despite this there is a reluctance to accept the possibility that identifica-
tion of peripheral biomarkers can be of any benefit. In addition, psychiatrists find
it difficult to accept that peripheral molecules such as blood-based proteins and
small molecules can reflect what is happening in the brain. However, the health
and regulatory authorities now consider that biomarkers are important for the
future of drug development and have called for efforts to modernize methods,
tools and techniques for this purpose. The authors describe the development of
the first ever molecular blood test for schizophrenia and the reactions of research
scientists and psychiatrists to this development, as a case in point. There is now
reason for optimism that further technological advancements and interdisciplin-
ary approaches in biomarker research will overcome current limitations and help
to advance our ability to treat patients with neuropsychiatric disorders.
The 13th chapter by Wong et al. addresses the need and movement toward
personalized medicine in the neuropsychiatric field. Advances in human genetics
and molecular innovations in neuroscience have prompted the pharmaceutical
industry to move beyond the treatment of broad spectrum diseases to more
targeted (personalized) treatment approaches. Recurring failure in converting
scientific discoveries in neuroscience to novel efficacious drugs has precipitated
a crisis in the industry. A targeted and consistent investment is needed to restore
confidence in translating science into clinical success. There are now movements
for cross-pharmaceutical company and globally coordinated efforts for discovery
of better, therapy-linked patient stratification, as exemplified by the European
Union Innovative Medicine Initiatives project entitled: ‘‘New Medications in
Depression and Schizophrenia—NEWMEDS.’’ That fact that such efforts are
now being made by individual pharmaceutical companies, suggests that the time
and opportunity for a fresh approach in this area are now welcome.
The 14th chapter by van Beveren and Hoogendijk covers the clinical aspects
of major neuropsychiatric disorders and the need for a paradigm shift to biomark-
er-assisted diagnostic tests. Thus far, the identification and application of such
biomarker tests have been sparse. This is likely to be due to the fact that the
existing diagnostic methods are based on long-standing heterogeneous concepts
in psychiatry. In addition, a shift to using biomarkers for conditions which have
been categorized for decades based on clinical phenomenology would not be
clinically useful. However, there is a pressing need for biomarkers which can be
used as an aid to the normal procedure to classify at-risk patients, such as young
people with prodromal symptoms for psychosis and existing patients who are
likely to progress to more severe states. The authors also stress that there is a need
for better classification of patient subtypes and to deconstruct the traditional
diagnoses in favor of using biomarker-assisted strategies to accomplish this.
The 15th chapter by Lowe describes the potential future of biomarkers in
neuropsychiatric diseases which may include developments in the areas of bio-
sensors, neuroinformatics, and e-neuropsychiatry. The emergence of molecular
xx PREFACE
Abstract
I. The Quest for Biomarkers in Neuroscience
A. Biomarkers in Clinical Practice
B. Biomarkers for the Development of Novel Therapeutics and in Basic Research
II. Tools for Biomarker Discovery in Neuroscience
III. Advancements in Biomarker Discovery in Neuroscience
A. Mouse Models
B. Human Data
C. Future Directions
IV. Considerations for Biomarker Discovery and Translation in Neuroscience
A. Disease Complexity
B. Sample Quality and Collection
C. Candidate Biomarker Validation
D. Systemic Approaches and Biomarker Initiatives
V. Outlook—The Perspective of Personalized Medicine
Acknowledgments
References
Abstract
The field of biomarker research has received increasing attention from both
the scientific community and funding organizations. According to the official
definition by the National Institutes of Health (NIH), ‘‘a biomarker is a charac-
teristic that is objectively measured and evaluated as an indicator of normal
biological processes, pathogenic processes, or pharmacological responses to a
therapeutic intervention’’ (Biomarkers Definitions Working Group et al., 2001).
The utilization of biomarkers for brain disorders is not a recent concept. In the
nineteenth century, Kraepelin established a writing scale to stratify patients suffering
from psychiatric disorders by measuring their writing pressure curves (Kraepelin,
1899). Due to the phenotypic heterogeneity and the lack of quantitative measures for
disease symptoms, biomarker discovery in the field of neuroscience has been con-
fronted with considerable challenges. This holds true especially for neuropsychiatric
disorders where, despite tremendous progress in understanding brain function, the
exact molecular underpinnings of mental dysfunction remain elusive. Because
biomarkers can differentiate between distinct biological states, their availability is
critical in clinical settings for premorbid diagnosis, patient stratification, and moni-
toring of disease progression and treatment. In this regard, established biomarkers in
other areas of medicine including human chorionic gonadotropin to determine
pregnancy (Spadoni et al., 1964), serum ferritin to measure anemia (Pasricha et al.,
2010), and cholesterol to predict cardiovascular disease risk (Kannel et al., 1979) have
significantly simplified clinical practice.
Currently, the diagnosis for all psychiatric disorders is symptomatic and relies
on interview-based communication between the patient and the physician. The
only means for disease categorization is the Diagnostic and Statistical Manual of Mental
Disorders (American Psychiatric Association, 2000). Although this manual may
thoroughly describe the symptomatology of different mental disorders, it does not
provide molecular correlates nor does it address the underlying disease etiology.
In addition to the lack of any measurable molecular entities, disease classification
is often confounded by symptomatic expressions because multiple psychiatric
disorders that exhibit similar indications can coexist (Turck et al., 2008).
GENERAL OVERVIEW: BIOMARKERS IN NEUROSCIENCE RESEARCH 3
‘‘dry’’ biomarkers, such as brain images (see Chapter ‘‘Imaging brain microglial
activation using positron emission tomography and translocator protein-specific
radioligands’’ by Owen and Matthews). Regardless of the biomarker discovery
platform type, appropriate quantitative assays need to be developed to translate
the experimental workflows into clinical practice.
The starting point of biomarker discovery for neuropsychiatric disorders is
either patient material or animal models that represent psychiatric endopheno-
types. Although human material is the most relevant specimen for analysis, the
interindividual variability together with the low sample amounts available can
pose serious challenges for analytical efforts. Not surprisingly, during the explor-
atory phase of the biomarker discovery pipeline, animal model-based studies tend
to have higher success rates compared to human-based studies due to the con-
trolled genetic background, the limited heterogeneity, and the large sample
cohorts that can be achieved in laboratory-bred animal populations (Turck
et al., 2005). However, because of the lack of defined lesions for most neuropsy-
chiatric conditions and the fact that the whole spectrum of neuropsychiatric
disorders in humans cannot be fully recapitulated in lower organisms, most of
the existing animal models aim to capture only specific disease characteristics or
endophenotypes (see Chapter ‘‘Behavioral and molecular biomarkers in transla-
tional animal models for neuropsychiatric disorders’’ by Sarnyai et al.). The study
of endophenotypes has provided useful insights into the psychopathology of
psychiatric disorders (Amann et al., 2010; Kendler and Neale, 2010; Puls and
Gallinat, 2008) and is a promising approach to identify biomarkers indicative of
disease progression or a given disease symptom. Nevertheless, care should be
taken when extrapolating conclusions drawn from animal models to humans.
The availability of -omics methods (genomics, transcriptomics, proteomics,
and metabolomics) and new powerful in vivo imaging technologies have improved
the understanding of psychiatric disorder pathophysiology by comprehensively
interrogating disease states at the molecular level. As a result of the development
of these holistic approaches, a shift from hypothesis-driven to hypothesis-free
studies has occurred, raising the possibility of identifying novel molecular entities
and affected brain circuits that constitute candidate biomarkers.
Genomic analyses have provided useful insights into genes conferring suscepti-
bility to complex neuropsychiatric diseases (Gill et al., 2010) as well as genes asso-
ciated with treatment resistance and efficacy (Binder et al., 2004; Foster et al., 2010;
Möller and Rujescu, 2010). Given that multiple genetic lesions, which may addi-
tionally vary among individuals, can cause psychiatric disorders, many disease-
related genes have a low penetrance and do not exhibit an effect on the phenotype
in a predictable and quantifiable manner (Schwarz and Bahn, 2008). This gap
between a genetic lesion and an effect on the behavioral phenotype can be bridged
by proteomics and metabolomics. Proteomic signatures are dynamic and have the
potential to reflect different disease states and reveal mechanisms of drug action
(Turck et al., 2008; see Chapter ‘‘Proteomic technologies for biomarker studies in
GENERAL OVERVIEW: BIOMARKERS IN NEUROSCIENCE RESEARCH 5
A. MOUSE MODELS
B. HUMAN DATA
3. Plasma Studies
Plasma constitutes the specimen of choice for the implementation of a bio-
marker assay in clinical settings. Despite its easy and noninvasive acquisition at
relatively high amounts from patients, plasma analysis presents researchers with
serious technical challenges, largely due to the great dynamic range of its protein
constituents ( Jacobs et al., 2005). Nevertheless, a multidisciplinary study in a large
cohort of well-characterized schizophrenic and major depressive patients has
resulted in identification of molecular candidate plasma biomarker signatures
(Domenici et al., 2010). Toward the clinical implementation of plasma biomarkers,
there have been attempts to provide molecular ‘‘kits’’ to aid in the diagnosis or risk
assessment for major psychiatric disorders. These mainly involve tests based on
genetic susceptibility to predict risk for developing a psychiatric disorder.
Although not yet commercially available, efforts to develop such tests are ongoing
(Couzin, 2008). Recently, a multiplex protein immunoassay-based plasma/serum
diagnostic test for schizophrenia was launched, assessing the levels of 51 molecules
for identification of patients with schizophrenia compared to healthy control
subjects (Schwarz et al., 2010, 2011; see Chapters ‘‘The application of multiplexed
assay systems for molecular diagnostics’’ by Schwarz et al. and ‘‘Algorithm
development for diagnostic biomarker assays’’ by Izmailov et al.). These kits
provide promising tools for the prognosis and classification of neuropsychiatric
disorders and constitute a first step toward biomarker-based molecular diagnos-
tics, indicating the potential of nonhypothesis-driven -omics approaches for bio-
marker discovery.
C. FUTURE DIRECTIONS
A. DISEASE COMPLEXITY
mirrored or are too low in abundance in plasma, which makes the validation of
biomarkers rather challenging. During the biomarker discovery phase, a well-
characterized, homogeneous patient cohort of limited size with adequately
matching controls is often used. In the validation phase though, larger cohorts
are studied where heterogeneity and intragroup-related variation are markedly
higher. This reduces the probability that a biomarker candidate will be verified,
minimizing its chances of qualifying for use in a clinical trial. Hence, large-scale
studies are required to ensure the specificity of a candidate biomarker and its
relevance to the disease under examination.
It is now widely accepted that a single biomarker will not be able to unequiv-
ocally distinguish between clinical neuropsychiatric phenotypes. A panel of bio-
markers that are able to depict a disease state more accurately is required for
complex diseases such as anxiety disorders, depression, and schizophrenia (Turck
et al., 2008). In this context, integrating -omics and imaging data will provide a
more comprehensive characterization of different disease states through a combi-
nation of biomarkers (e.g., altered protein levels and functional magnetic reso-
nance imaging (fMRI) profiles combined with genetic information), which in turn
will enable more accurate disease diagnoses and patient-specific treatment
options. Currently, no molecular biomarker is available for neuropsychiatric
disorders, and there is still a long path ahead toward clinical implementation.
However, there are reasons for optimism that further technological advancements
and interdisciplinary approaches will overcome the current limitations in the field
and will finally enable the concept of personalized medicine (see Chapter
‘‘Toward personalized medicine in the neuropsychiatric field’’ by Wong et al.).
Acknowledgments
The authors would like to thank all members of the ‘Proteomics and Biomar-
kers Research Group’ at the Max Planck Institute of Psychiatry for insightful
discussions.
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IMAGING BRAIN MICROGLIAL ACTIVATION USING
POSITRON EMISSION TOMOGRAPHY AND TRANSLOCATOR
PROTEIN-SPECIFIC RADIOLIGANDS
Abstract
I.Introduction
II.Principles of PET Imaging
III.TSPO for Assessment of Microglial Expression
IV. Challenges Facing PET Imaging of the TSPO
V. Disease Applications
A. Neuroinflammatory Diseases
B. Neurodegenerative Diseases
C. Movement Disorders
D. Stroke
E. Neuropsychiatric Diseases
VI. Conclusion
Acknowledgments
References
Abstract
I. Introduction
PET imaging studies require the design of a ligand which binds with high
specificity to a desired target, but with minimal nonspecific binding to other
structures. The ligand is labeled with a positron emitting radioisotope with a
short half-life (t1\2), commonly 11C (t1\2 20 min) or 18F (t1\2 110 min).
Following intravenous administration of the radiolabeled ligand (radioligand),
IMAGING BRAIN MICROGLIAL ACTIVATION USING POSITRON EMISSION TOMOGRAPHY 21
the emitted positrons will collide with nearby electrons resulting in the production
of pairs of photons that travel at 180 to each other. The photon pairs are detected
by g-detectors surrounding the subject, allowing the spatial distribution of the
radioligand to be reconstructed, once corrections have been made for scatter,
attenuation, random detection events, dead time, and detector efficiencies.
To relate spatial distribution to anatomy, PET images are coregistered
with structural images from computed tomography (CT) or magnetic resonance
imaging (MRI) images. The effective spatial resolution of a PET scan is approxi-
mately 4 mm.
Development of useful, new PET radioligands, however, is nontrivial.
A sufficiently specific molecule target must be chosen. The affinity at which the
radioligand binds to the target must be sufficiently high to produce a detectable
signal, but the radioligand must dissociate from the target quickly enough to allow
the binding equilibrium to be approached within the timeframe of the scan
(1–2 h). Lipid solubility is required to allow the radioligand to cross the blood
brain barrier (BBB), but radioligands which are too lipid soluble will bind non-
specifically to cell membranes and produce a high background signal (Guo et al.,
2009). Radioligands can be rapidly metabolized following administration. If the
metabolites also are radiolabeled and can cross the BBB, the resultant combined
signal can be too complex to interpret quantitatively. Finally, synthesis of the
radioligand must be simple enough to be performed rapidly (and to pharmaceu-
tical standards), given the short half-lives of the isotopes.
Various methods can be used to derive quantitative data for PET. Clinical
PET scans, such as 18F-fluorodeoxyglucose (FDG) scans to detect tumors, are
usually reported based on the signal to background contrast obtained from a
single three-dimensional image. The standardized uptake value (SUV) is a crude
means of signal quantification, calculated by normalizing the signal measured by
the PET camera within the region of interest to the injected dose and body weight
(Thie, 2004). However, SUVs do not distinguish between molecules of the radi-
oligand which are specifically bound to the target (which is the measurement of
interest) compared to the radioligand molecules which are bound nonspecifically
to other structures or unbound in the blood or tissue.
More complex analyses, based on kinetic modeling of the dynamically
acquired signal over time, allow for the component of the signal which represents
specific binding of the radioligand to the target to be extracted. The modeling is
simplified when there is a ‘‘reference region’’ within the brain which is devoid of
the target. Such reference regions are useful for the determination of nonspecific
binding. In cases where targets are expressed throughout the brain and therefore
do not have a reference region (such as with the TSPO), more complex
approaches are needed to estimate the nonspecific binding (Turkheimer
et al., 2007).
22 DAVID R.J. OWEN AND PAUL M. MATTHEWS
et al., 2007), [11C]DPA-713 (Endres et al., 2009), [18F]PBR111 (Fookes et al., 2008;
Van et al., 2010), and [11C]PBR28 (Imaizumi et al., 2008).
Recent clinical PET studies using these radioligands have confirmed that they
have substantially improved SBR compared to that with [11C]PK11195, but
revealed an unexpected complication to their use. With the [11C]PBR28 ligand,
approximately 10% of human subjects do not show specific TSPO binding
(Venneti et al., 2008). We have recently clarified that such subjects express the
TSPO, but that it shows an approximate 50-fold reduction in affinity for PBR28.
We have called such subjects low-affinity binders (LABs; Owen et al., 2010).
Among those remaining subjects with a measurable specific PET signal using
[11C]PBR28, two further groups can be identified: high-affinity binders (HABs),
who express a single class of high-affinity binding sites, and mixed-affinity binders
(MABs), who express approximately equal proportions of high- and low-affinity
binding sites (Fig. 1). This phenomenon is not limited to [11C]PBR28 as we have
also shown that all new generation TSPO tracers recognize these three binding
classes (Owen et al., 2011). The reason this may have gone undetected with these
150
100
150
50
Percentage of specific binding
100
0 2 4
Concentration of unlabeled
PBR28 (log nM)
50
–2 0 2 4
Concentration of unlabeled PBR28 (log nM)
FIG. 1. Radioligand binding competition assay in human brain tissue with [3H]PK11195 in the
presence of increasing concentrations of unlabeled PBR28. Curve fit is shown for each sample
(n ¼ 15). Inset—one representative curve with data points for each binding category. Black, low-
affinity binder; Red, high-affinity binder; Green, mixed-affinity binder.
IMAGING BRAIN MICROGLIAL ACTIVATION USING POSITRON EMISSION TOMOGRAPHY 25
tracers hitherto is that, with the exception of PBR28, the reduction in affinity with
LABs compared to HABs is relatively small (four- to sixfold). Crucially, however,
in such studies the PET signal will substantially underestimate TSPO expression
in subjects who express the low-affinity receptor (namely LABs and MABs).
Complicating matters further is the fact that binding affinity in the brain cannot
be ascertained from a single PET scan. We have shown, however, that measuring
binding affinity in platelets isolated from whole blood is feasible and that the HAB,
MAB, and LAB binding patterns seen in the brain are also present in platelets
(Owens, R.D.J., Matthews, P.M., Rabiner, E.A., Parker, C.A., and Gunn, R.N.,
unpublished findings). Platelet binding data could therefore potentially be used to
determine a subject’s TSPO-binding affinity. With this information, the binding
potential measurements obtained with PET could be corrected to allow valid
comparisons between subjects of different binding classes. However, further work
is first required to confirm the correspondence of platelet and brain binding affinities.
V. Disease Applications
A. NEUROINFLAMMATORY DISEASES
A B C D
FIG. 2. TSPO distribution in an acute demyelinating lesion in postmortem brain tissue from a
subject with MS. (A) Binding with TSPO ligand [3H]PBR28 showing high signal (red arrow) around
the periphery of the lesion and lower signal in normal white (white arrow) and gray (black arrow)
matter. (B) Binding of neighboring section with TSPO ligand [3H]PBR28 in the presence of an excess
of unlabeled PK11195. Because almost all TSPO is bound to unlabeled PK11195, no signal from [3H]
PBR28 is evident demonstrating the specificity of [3H]PBR28. (C) Low MHC II expression from
normal-appearing white matter (400) colocalizing with low [3H]PBR28 binding (e.g., white arrow).
(D) High MHC II expression from white matter lesion (400) corresponding with high [3H]PBR28
binding (e.g., red arrow).
FIG. 3. Serial scanning in multiple sclerosis patients showed that the [11C]PBR28 signal can
precede the appearance of MRI contrast enhancement, suggesting that glial activation may be an
early event in MS lesion formation. Postcontrast T1-weighted MRI (left) and coregistered [11C]PBR28
VT difference map (center). Gadolinium enhancement of the same region is detected a month later
(right; Oh et al., 2010).
however, although a significant but weak correlation was found between global
[11C]PBR28 binding levels and disease duration, global binding did not differ
significantly between groups of healthy volunteers and subjects with MS (Oh et al.,
2010). This deserves further study.
B. NEURODEGENERATIVE DISEASES
signal in temporal and parietal lobe, TSPO binding was found to colocalize
with immunohistochemical microglial markers (Gulyas et al., 2009).
PET data also has been encouraging. The first report of in vivo microglial
activation in AD was in 2001, in which [11C]PK11195 binding was significantly
increased relative to that in control subjects, and the spatial distribution of
increased binding was consistent with the characteristic pattern of AD. Interest-
ingly, binding was also increased in a subject with mild cognitive impairment
(MCI), suggesting that microglial activation may be detectable prior to the
development of overt AD. However, there was substantial overlap in signal
between the AD group and controls which would severely limit the utility of the
scan in clinical practice (Cagnin et al., 2001).
Recent studies using both [11C]PK11195 to image microglia and [11C]PIB to
assess amyloid load have confirmed an increase in the [11C]PK11195 signal in
several cortical regions in patients with AD (Edison et al., 2008; Yokokura et al.,
2011). In these studies, there was no correlation between microglial activation and
amyloid load (measured as the [11C]PIB signal), suggesting that these processes
are not linked in a simple way. In both studies, the dementia score was inversely
correlated with [11C]PK11195, but not with the [11C]PIB signal, suggesting that
microglial activation becomes more pronounced as the dementia progresses. This
is consistent with data from studies of subjects with MCI (Okello et al., 2009). It
may also explain why one recent study found no difference in the [11C]PK11195
signal between AD, MCI, and control subjects, as the AD subjects in this study
had mild disease only (Wiley et al., 2009).
The relatively small changes in [11C]PK11195 signal between control, MCI,
and mild AD subjects may be difficult to discriminate, given the poor SBR of the
[11C]PK11195 tracer. Studies using new generation TSPO ligands, with signifi-
cantly enhanced SNR profiles, are likely to help clarify the situation and may
show greater separation between the two groups. Although such a study has been
performed in AD and controls subjects with [11C]DAA1106, this study did not
take into account the effect of variable binding affinity with new generation
ligands (Owen et al., 2010, 2011). Therefore, although a significant increase in
signal in the AD group was detected, there was substantial overlap in the signal
between the two groups (Yasuno et al., 2008).
The lack of correlation between amyloid deposition and microglial activation
in AD suggests that other forms of dementia might also be characterized by
increased TSPO binding. Indeed, as with AD, autoradiography studies of postmor-
tem brains have shown that in frontotemporal demential (FTD), TSPO density is
elevated in affected regions compared to that seen in controls (Venneti et al., 2008).
Further, increased binding was also seen in a PET study with [11C]DAA1106 in
three subjects with no neurologic abnormalities, but who had been diagnosed
with preclinical FTD based on their genotype (Miyoshi et al., 2010).
IMAGING BRAIN MICROGLIAL ACTIVATION USING POSITRON EMISSION TOMOGRAPHY 29
C. MOVEMENT DISORDERS
1. Parkinson’s Disease
The rationale for using TSPO-PET analyses to quantify neuroinflammation
in Parkinson’s disease (PD) is based on immunohistochemical data showing large
numbers of microglia within the substantia nigra region of postmortem brain
samples from subjects with PD. By contrast, control subjects have few microglia
in the substantia nigra (Banati et al., 1998; McGeer et al., 1988). Interestingly, the
microglial activation in these studies appears to be independent of disease dura-
tion and severity (Banati et al., 1998). Studies in patients with PD plus syndromes
have demonstrated the feasibility of imaging the basal ganglia with the [11C]
PK11195 tracer in multisystem atrophy (Gerhard et al., 2003), progressive supra-
nuclear palsy (Gerhard et al., 2006a,b), and corticobasal degeneration (Gerhard
et al., 2004; Henkel et al., 2004). [11C]PK11195 PET analyses revealed patterns of
increased microglial activation that corresponds well with the known distribution
of microglia in these diseases.
Two PET studies with [11C]PK11195 have together confirmed that quantify-
ing microglial activation in vivo in patients with PD is possible (Gerhard et al.,
2006a,b; Ouchi et al., 2005). Ouchi et al. studied drug-naive patients with mild
disease and reported an increased [11C]PK11195 signal in the midbrain in
patients compared to the signal in controls. Although the [11C]PK11195 signal
did not correlate with disease duration, there were positive correlations found
with measures of severity of motor deficits and loss of dopaminergic projections in
the putamen region of the dorsal striatum (Ouchi et al., 2005). Gerhard et al.
studied patients with more severe disease and found more global changes includ-
ing an increased [11C]PK11195 signal in the striatum, pallidum, and pons, as well
as in cortical areas (Gerhard et al., 2006a,b). However, a correlation neither
between signal and duration of disease nor between [11C]PK11195 signal and
disease severity or loss of dopaminergic projections was found. The independence
of the microglial TSPO signal and clinical symptoms was further confirmed by
follow-up scans approximately 2 years later in a subset of the study population. No
change in [11C]PK11195 signal was observed, despite clinical deterioration and a
further degeneration of dopaminergic neurons. A recent pilot study assessing the
pharmacodynamic effects of anti-inflammatory treatment in PD also showed no
significant differences at baseline between groups of PD patients (both drug naive
and advanced disease) and controls (Bartels et al., 2010).
The reasons underlying the discrepancies between these three studies are not
clear. It is significant that the results within each study varied greatly with different
methods of image analysis, highlighting the difficulties in accurately quantifying
[11C]PK11195 binding in vivo. Theoretically, based on the fact that substantial
microglial activation is known to occur in PD, TSPO-PET analysis should be a
30 DAVID R.J. OWEN AND PAUL M. MATTHEWS
2. Huntington’s Disease
In HD, reactive microglia are present at all stages and accumulate with
severity of disease in proportion to the degree of neuronal loss (Sapp et al.,
2001). TSPO-binding sites are increased in the HD brain, with a 70% increase
in binding in the putamen, a 25% increase in the frontal lobe, and a 10% increase
in the temporal lobe. A first TSPO-PET study in HD found increased [11C]
PK11195 binding in the striatum and cortex of patients relative to that in healthy
controls. Further, the binding correlated with clinical severity as assessed by motor
scores and correlated inversely with striated [11C]raclopride binding, a measure
dopamine receptor density that provides an index of striated neurodegeneration.
A voxel-based analysis suggested colocalization of the increased [11C]PK11195
binding with the decreased [11C]raclopride binding (Pavese et al., 2006). A study
assessing presymptomatic HD gene carriers found a similar increase in [11C]
PK11195 binding and colocalization with decreased [11C]raclopride binding,
suggesting that microglial activation is an early event in the evolution of disease
pathology (Tai et al., 2007).
D. STROKE
Microglia are activated by neuronal injury and death and a robust microglial
response follows cerebrovascular infarction (Wang et al., 2007; Wood, 1995).
Large increases in monocyte-derived cells are found in the ischemic core within
48 h of infarction (Krupinski et al., 1996; Tomimoto et al., 1996) and there is an
approximate threefold increase in TSPO binding in postmortem human brain
samples in the core of infarcts (Venneti et al., 2008).
Most evidence suggests that activated microglial cells may contribute to injury
following infarction by release of proinflammatory cytokines and directly by
cytotoxic molecules. In a rodent model of ischemia, inhibition of microglial
activation with minocycline has a neuroprotective effect (Yrjanheikki et al.,
1998, 1999). Likewise, in neuron and oligodendrocyte cultures, microglia/macro-
phages are associated with greater injury during ischemia (Giulian et al., 1993;
Lehnardt et al., 2003; Zhang et al., 1997). However, other studies provide unequiv-
ocal evidence that microglia/macrophages, or their secreted factors, can also
protect cells against neuronal damage (Watanabe et al., 2000). The extent to
which the microglial response following brain ischemia is deleterious, or only in
IMAGING BRAIN MICROGLIAL ACTIVATION USING POSITRON EMISSION TOMOGRAPHY 31
part beneficial, is currently not clear (Wang et al., 2007). Microglial phenotypes
may change with time or may be spatially heterogeneous within lesions. Further,
like macrophages, microglia differ phentotypically and it is likely that different
phenotypes may have different effects on the resolution of lesions.
Two patterns of microglial activation have been observed following photo-
chemically induced focal ischemia of the rat cortex (Schroeter et al., 1999).
Initially, phagocytic microglia expressing major histocompatibility (MHC)
class I receptors are present in the core and periphery of the ischemic lesion.
Subsequently, after a delay of days, microglia with low phagocytic activity but with
high MHC Class II expression appear along degenerating fiber tracts with
connections to the infarct. This remote activation of microglia is thought to
represent Wallerian degeneration as a consequence of focal damage.
Evidence consistent with both patterns of microglial activation was reported
with the first use of the [11C]PK11195 ligand to measure TSPO expression
following cerebral ischemia in humans (Ramsay et al., 1992). In this case, a single
patient was studied following ischemic stroke. An increased signal was identified
adjacent to the lesion 6 days following stroke. When the patient was imaged again
1 week later, there was also evidence of tracer binding in areas remote from the
lesion. Similar findings were reported in a study of a series of seven patients with
middle cerebral artery infarcts. All patients showed increased [11C]PK11195
binding in the thalamus ipsilateral to the infarct, that persisted at 24 months of
follow-up (Pappata et al., 2000).
The kinetics of the signal change was investigated in a small series of patients
scanned between 3 and 150 days following cerebral ischemia. Increased [11C]
PK11195 binding around the lesion was observed as early as 3 days after
ischemia. Subsequently, both the primary lesion and areas distant from the
primary lesion site began to show increases in signal (Gerhard et al., 2005). In
another series, signal changes were examined over three time points (< 72 h, week
2, week 4). In this study, minimal binding was found within 72 h in the core of the
lesion, but binding rose significantly by week 2 before reducing slightly by week 4.
As with previous studies, binding was present from early on in the core and
periphery of the lesion and extended to remote zones after 7–10 days (Price
et al., 2006).
A recent study tested the hypothesis that signal changes in areas remote to the
lesion are a direct response to the ischemic insult. The [11C]PK11195 signal was
measured in the pyramidal tract (PT) of patients with acute subcortical ischemia,
and an increased signal was only found in patients in whom the PT was affected
by stroke. In stroke patients in which the lesioned brain area did not project to the
PT, no signal change in the PT was detected (Radlinska et al., 2009). In a similar
experiment in which patients were investigated serially, the same authors found
that while microglial activity in the infarct returns to baseline levels with time,
microglia in the remote regions persisted throughout the 6-month follow-up
32 DAVID R.J. OWEN AND PAUL M. MATTHEWS
period. Although greater microglial activation in the lesion was correlated with
poor clinical outcome, remote microglial activation was correlated with improved
clinical outcome (Thiel et al., 2010).
E. NEUROPSYCHIATRIC DISEASES
VI. Conclusion
Acknowledgments
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THE UTILITY OF GENE EXPRESSION IN BLOOD CELLS FOR
DIAGNOSING NEUROPSYCHIATRIC DISORDERS
Abstract
I. Introduction
II. Microarray Gene Expression Analysis
III. Diagnostic Gene Expression Classifiers
IV. Blood Gene Expression Studies of Neuropsychiatric Disorders
V. MicroRNA Expression Analysis
VI. Pharmacogenomics
VII. Concluding Remarks
Acknowledgments
References
Abstract
I. Introduction
There are currently no objective genetic tests in widespread use for the
diagnosis of any one of a large number of neuropsychiatric disorders. The exact
pathogenesis of most neuropsychiatric disorders remains unclear with a promi-
nent role for biological, genetic, and environmental factors (Glatt et al., 2010).
Subjective clinical evaluations of disorders such as schizophrenia, bipolar disor-
der, Parkinson’s disease, posttraumatic stress disorder (PTSD), and major depres-
sive disorder (MDD) may sometimes lead to inconsistencies in diagnosis between
different psychiatrists especially when patients are followed longitudinally. Gene
expression analysis of the entire transcriptome using microarray technology is
now a common laboratory practice with demonstrated reproducibility (Shi et al.,
2006). This chapter investigates the feasibility of using gene expression to con-
struct objective classifiers for the diagnosis of neuropsychiatric disorders.
The use of gene expression classifiers for disease prognosis has already met
with approval from the United States Food and Drug Administration (FDA).
In 2007, the FDA granted approval to MammaPrintÒ, a classifier constructed
from the expression of 70 genes in tumors biopsied from women with breast
cancer that is capable of predicting recurrence within 5 years (vant Veer et al.,
2002). MammaPrintÒ is currently being evaluated in the MINDACT (Microarray
in Node-Negative Disease May Avoid Chemotherapy) trial to assess its utility for
identifying patients with a low risk of recurrence, and who can therefore be spared
the inconvenience and morbidity associated with adjuvant chemotherapy
(Cardoso et al., 2008). MammaPrintÒ and future gene expression classifiers are
defined by the FDA as in vitro diagnostic multivariate index assays (IVDMIAs)
which require a complex algorithm to calculate the diagnostic score. This is in
contrast to traditional biomarkers for which a simple concentration (i.e., blood
cholesterol) is used as a score for diagnosis (Kato, 2009; see Chapter ‘‘Algorithm
development for diagnostic biomarker assays’’ by Izmailov et al.).
Due to its accessibility, peripheral blood has historically been a valuable tissue
source for the derivation of biomarkers (Woelk and Burczynski, 2008). A plethora
of studies have been published recently demonstrating the utility of constructing
gene expression classifiers from blood cells in order to diagnose disease states,
predict disease outcomes, and determine an individual’s response to treatment
THE UTILITY OF GENE EXPRESSION IN BLOOD CELLS 43
(Burczynski and Dorner, 2006; Woelk and Burczynski, 2008). With respect to
neuropsychiatric disorders, it is important to define the difference between a
mechanistic- and a classifier-based gene expression study. Mechanistic studies
have focused on analyzing gene expression in brain tissue, which, in the case of
schizophrenia, is normally derived from the prefrontal cortex (Garbett et al., 2008;
Glatt et al., 2005; Maycox et al., 2009; Narayan et al., 2008; Prabakaran et al.,
2004), although other brain regions have also been analyzed, that is, the superior
temporal gyrus (Bowden et al., 2008). Methods of class comparison are then used
in order to identify differentially expressed genes that are mapped on to biological
pathways, gene ontologies, and protein networks in order to reveal the higher-
level processes contributing to disease pathogenesis. In a classifier study, methods
of class prediction are used to identify those genes whose expression can be used to
predict whether a blinded sample came from an individual with a neuropsychia-
tric disorder or a healthy control. Obviously, sampling brain tissue for such a
diagnostic purpose is not feasible (Struyf et al., 2008) and many classifier studies
have focused on analyzing gene expression in blood cells for this purpose (Table I).
Our own studies, among others, have focused on the overlap in gene expression
between the blood and brain compartments in order to evaluate the utility of gene
expression in the blood for diagnosing neuropsychiatric disorders (Glatt et al., 2005;
Shehadeh et al., 2010; Sullivan et al., 2006). For example, we previously identified
177 genes differentially expressed in the dorsolateral prefrontal cortex (DLPFC)
between 19 schizophrenia and 27 control cases (Glatt et al., 2005). When gene
expression was analyzed in white blood cells (WBCs) derived from 30 schizophre-
nia and 24 control cases in an unrelated cohort, 123 genes were identified as being
differentially expressed. An overlap of only two genes modulated in the same
manner was found between compartments. The level of selenium binding protein
1 (SELENBP1) mRNA was increased, and that of major histocompatibility com-
plex, class II, DR beta 1 (HLA-DRB1) was decreased, in both compartments. The
differential expression of SELENBP1 was confirmed at the mRNA transcript level
by real-time quantitative PCR (RT-qPCR) and at the protein level by antibody
staining. In addition, this finding was subsequently replicated in brain tissue
samples from an independent cohort (Kanazawa et al., 2008). Although only a
small overlap in differential gene expression between schizophrenia and control
cases across compartments was revealed in our study, it should not be taken as
evidence that the blood compartment is of limited use for the derivation of
diagnostic gene expression signatures. The prevailing hypothesis behind hemoge-
nomic studies that attempt to construct gene expression classifiers is that a tran-
scriptional signature exists in common to all neuropsychiatric cases that is distinct
from all control samples (Woelk and Burczynski, 2008). If this is the case, then it is
of no consequence whether the genes whose expression comprises the signature
are expressed in brain tissue; they are simply being used as a diagnostic read out
and not intended for mechanistic interpretation (Middleton et al., 2005).
Table I
BLOOD GENE EXPRESSION STUDIES OF NEUROPSYCHIATRIC DISORDERS THAT DEVELOPED A DIAGNOSTIC CLASSIFIER.a
Disorder Tissue source Number of microarrays Microarray Classifier Validation Classifier GEO Reference
platform method performanceb
Total Disorder Drug- Control
naive
Bipolar disorder Whole blood 78 37 (HM), 13 0 NA Affymetrix HG Mood prediction Hold-out PD test data set: No Le-Niculescu
and psychotic (IM), and U133 Plus 2.0 score HM—71.4% et al. (2009)
disorders 28 (LM) GeneChip sensitivity, 62.5%
specificity; LM—
66.7% sensitivity,
61.9% specificity
BPD test data set:
HM—70%
sensitivity, 66.7%
specificity; LM—
66.7% sensitivity,
61.5% specificity
Major depressive Whole blood 67 33 33 34 Agilent 44K Nearest Hold-out 74.1% GSE19738 Spijker et al.
disorder (LPS- Human shrunken (2010)
stimulated) whole centroid and
genome array MDD score
Posttraumatic PBMCs 33 17 NR 16 Affymetrix Naive Bayesian LOOCV 89% for M4 samplesc GDS1020 Segman et al.
stress disorder HU95A (2005)
GeneChip
Schizophrenia Whole blood 101 52 44 49 CodeLink Artificial neural Threefold CV 91.2%—training No Takahashi et al.
Human network and hold-out data set 87.9%— (2010)
Whole test data set
Genome
Bioarray
Schizophrenia WBCs 76 33 (SZ) and 5 NR 38 Affymetrix HG Support vector Hold-out 100% No Middleton et al.
and bipolar (BPD) U133A machine (2005)
disorder GeneChip
WBCs 54 30 (SZ) and 7 0 17 Affymetrix HG Logistic NAd 0.960 (AUC for SZ) No Tsuang et al.
(BPD) U133A and regression 0.948 (AUC for (2005)
U133Plus 2.0 BPD)
GeneChip
a
Drug-naive is a subset of the subjects in the Disorder column and refers to the number of samples used for microarray analysis that were taken from patients with the disorder
that were also drug-naive or drug-free. Shading is for display purposes only. Abbreviations: GEO, refers to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and
whether microarray data has been deposited in this public repository; Ref., refers to the literature reference for the microarray study; HM, high mood; IM, intermediate mood;
LM, low mood; NR, not reported; NA, not applicable; PD, psychotic disorder; BPD, bipolar disorder; LPS, lipopolysaccharide; MDD, major depressive disorder; PBMCs,
peripheral blood mononuclear cells; LOOCV, leave-one-out cross-validation; WBCs, white blood cells; SZ, schizophrenia; AUC, area under the curve from receiver operator
curve analysis.
b
Classification accuracy is presented unless otherwise stated.
c
M4 samples were those taken 4 months after a traumatic event (N ¼ 9).
d
Analysis of microarray gene expression data was used to construct an eight gene classifier whose expression was then evaluated by RT-qPCR in some of the same samples used
for microarray analysis. AUC values are given for the performance of this RT-qPCR classifier, which was not validated in a k-fold cross-validation or hold-out validation
procedure.
46 CHRISTOPHER H. WOELK ET AL.
The typical workflow for a microarray gene expression study that aims to
construct a diagnostic gene expression classifier is outlined in Fig. 1. Previous gene
expression studies have analyzed many different subsets of cells from the blood
Whole blood,
WBCs,
PBMCs, Sample selection
lymphocytes,
monocytes
Sample size
Power analysis
calculator
e.g. Fisher’s exact
test or chi-square Case versus control
test for gender group statistics
differences
e.g. Qiagen
Isolate RNA
miRNeasy Mini Kit
FIG. 1. A typical workflow for a microarray gene expression study aiming to construct a diagnostic
gene expression classifier. Sample size calculator refers to the method developed by Dobbin et al. (2008)
available online (http://linus.nci.nih.gov/brb/samplesize/samplesize4GE.html). Abbreviations: MA, log-
intensity M-values versus log-intensity averages or A-values; WBCs, white blood cells; PBMCs, peripheral
blood mononuclear cells; NUSE, normalized unscaled standard error; RLE, relative log expression.
THE UTILITY OF GENE EXPRESSION IN BLOOD CELLS 47
Number of microarrays
Disorder Tissue source Total Disorder Drug-naive Control Microarray platform GEO Ref.
Parkinson’s disease Whole blood 105 50 NRb 55 Affymetrix HG U133A GeneChip GSE6613 Scherzer et al. (2007)
Posttraumatic stress Monocytes 67 34 34 33 CodeLink Human Whole Genome No Neylan et al. (2010)
disorder Bioarray
Whole blood 35 15 15 20 Affymetrix HG U133 Plus 2.0 GeneChip No Yehuda et al. (2009)
Whole blood 16 8 8 8 Stress/immune chips (custom cDNA No Zieker et al. (2007)
array)
Schizophrenia WBCs 54 30 0 24 Affymetrix HG U133A GeneChip No Glatt et al. (2005)
Whole blood 64 32 32 32 Affymetrix HG U133 Plus 2.0 GeneChip No Kuzman et al. (2009)
LCL 15 8 7 7 Human Genome v.2.1 oligo arrays No Matigian et al. (2008)
(custom cDNA array)
LCL 14 5 NR 9 Research Genetics NIA-Neuroarray No Vawter et al. (2004)
(custom cDNA array)
Lymphocytes 23 13 13 10 Full Moon BioSystems cDNA slides No Zvara et al. (2005)
(custom cDNA array)
Schizophrenia and PBMCs 71 47 1 24 Affymetrix Human Exon 1.0 ST No Bousman et al. (2010)
bipolar disorder GeneChip and Affymetrix HG U133A
or U133Plus 2.0 GeneChip
a
Column headings, table descriptions, and abbreviations are defined as for Table I except for LCL, which refers to lymphoblastoid cell line. Shading is for
display purposes only.
b
Scherzer et al. (2007) did not indicate if the same Parkinson’s disease patient was treated with more than one therapy so the exact number of drug-naive or
drug-free individuals could not be determined. For the 50 Parkinson’s disease patients analyzed in the study, 31 were treated with L-dopa, 24 with dopamine
agonist, and seven with selegiline.
THE UTILITY OF GENE EXPRESSION IN BLOOD CELLS 49
microarray gene expression study has been published, the data should be depos-
ited in a public repository such as ArrayExpress (http://www.ebi.ac.uk/
arrayexpress/) at the European Bioinformatics Institute or the Gene Expression
Omnibus (http://www.ncbi.nlm.nih.gov/geo/) at the National Center for Bio-
technology Information. This facilitates reanalysis of the microarray data with
novel methods as they emerge and also allows the merging of studies to facilitate
meta-analyses with increased power. Discouragingly, the vast majority (13/16) of
neuropsychiatric studies assessing gene expression in blood cells have not deposited
their microarray data in public repositories (Tables I and II).
Many useful tools have been developed for the construction of gene expression
classifiers and those that are freely available include BRB-Array Tools (Simon
et al., 2007), MCRestimate (Ruschhaupt et al., 2004), and Babelomics (Al-Shahrour
et al., 2008). Each of these tools use methods of class prediction: a supervised
approach that incorporates the sample label (e.g., schizophrenia or control) to
identify the genes whose expression can be used to predict which group a blinded
sample belongs to. Multiple methods have been used to classify samples and
include support vector machines (SVMs), artificial neural networks (ANNs),
logistic regression, and Bayesian methods (Middleton et al., 2005; Segman et al.,
2005; Takahashi et al., 2010; Tsuang et al., 2005).
In order to evaluate classifier performance, a 2 2 contingency table needs
to be populated so that the numbers of true positives (TP), true negatives (TN),
false positives (FP), and false negatives (FN) may be estimated in order to calculate
classifier accuracy (TP þ TN/TP þ TN þ FP þ FN), sensitivity (TP/TP þ
FN), and specificity (TN/TN þ FP). A number of strategies exist for populating
such a 2 2 contingency table but the most common are hold-out and k-fold
cross-validation (Dupuy and Simon, 2007). In hold-out validation, a number of
samples are removed from analysis as an independent test data set and the
remaining samples used to construct and train the gene expression classifier.
Then the 2 2 contingency table is populated by predicting the class of the
samples in the test data set using the classifier constructed using the training data
set. Hold-out validation is normally used when a large number of samples are
available (Le-Niculescu et al., 2009; Middleton et al., 2005; Takahashi et al., 1996),
whereas most smaller pilot studies employ k-fold (commonly 1 or 10) cross-
validation to assess classifier accuracy. In the case of leave-one-out cross-validation
(LOOCV), a sample is removed from the training data, the remaining samples
used to construct the classifier, and then this classifier is used to predict the class of
THE UTILITY OF GENE EXPRESSION IN BLOOD CELLS 51
the sample that was left out. This process is repeated until every sample has been
left out at least once and the predictions of each sample are used to populate the
2 2 contingency table. In both these validation procedures, a set of genes must
be selected whose expression is used for classification purposes. This set of genes is
sometimes selected as all those with a p-value above a certain threshold but more
commonly identified using a method of feature selection. For example, in BRB-
ArrayTools it is possible to perform recursive feature elimination where the
number of genes desired in the classifier is selected a priori and then a machine
learning method (i.e., SVM) is used to remove genes from the classifier based on
their lack of contribution to prediction performance (Simon et al., 2009).
There are several essential steps that are required in order to construct a
diagnostic gene expression classifier with clinical utility for neuropsychiatric
studies. These steps include benefit, feasibility, internal validation, external vali-
dation, and a prospective trial. Benefit is an initial assessment of whether a
diagnostic gene expression classifier is likely to improve on the accuracy or reduce
the cost of any diagnostic tool that already exists for the disease. In the case of
neuropsychiatric disorders, there are currently no diagnostic genetic tests in
widespread use and clinical diagnosis requires a battery of tests incurring signifi-
cant costs and substantial amounts of clinical time. Therefore, there is clearly a
great need for diagnostic gene expression classifiers for neuropsychiatric disor-
ders. Feasibility refers to a pilot study whereby the utility of measuring gene
expression in the blood for diagnosing the neuropsychiatric disorder under
study is established. A larger number of patients from the same cohort that was
used in the pilot study should then be analyzed in the internal validation step. Once a
diagnostic gene expression classifier has been developed, it needs to be subjected
to external validation in an unrelated cohort. For example, if a diagnostic gene
expression classifier was constructed using samples taken from schizophrenia
and control cases in Japan (Takahashi et al., 2010), the accuracy of the classifier
would then need to be assessed for a similar cohort in the United States or Europe.
Finally, a prospective trial should be initiated to evaluate the clinical utility of a
diagnostic gene expression classifier for the neuropsychiatric disorder. Patients
could be diagnosed with both a gene expression classifier and by a large panel of
trained psychiatrists. When discordant diagnoses arise, the patients could then be
followed up longitudinally to determine whether the classifier or the traditional
diagnostic approach was more accurate. The genes whose expression are being
used for classification purposes can be transferred onto a different platform, for
example, a custom miniature microarray or an RT-qPCR platform (i.e.,
TaqManÒ low density arrays, Applied Biosystems, Carlsbad, CA, USA). Howev-
er, this new platform would need to be internally and externally validated;
therefore there is an argument for performing this step earlier in the process of
developing a diagnostic gene expression classifier (Spijker et al., 2010; Tsuang et al.,
2005). All of the studies using blood gene expression to diagnose neuropsychiatric
52 CHRISTOPHER H. WOELK ET AL.
A large number of studies have been performed that analyzed gene expression
in blood cells derived from patients with neuropsychiatric disorders (Tables I and
II). A subset of these studies have demonstrated the feasibility of using blood gene
expression for diagnosing neuropsychiatric disorders and constructed classifiers of
moderate to high accuracy (Table I). A potential obstacle to deriving a diagnostic
gene expression classifier for neuropsychiatric disorders is the availability of drug-
naive and drug-free patients. This does not appear to be a problem when
analyzing data from PTSD cohorts but is a major confounder for schizophrenia
studies. Blood gene expression studies have primarily focused on schizophrenia
(9/16) where the majority of patients are often undergoing drug treatment or
treatment status is not reported (Tables I and II). Therefore, when classifiers are
constructed from such cohorts, it is unclear as to whether they are diagnosing
disease status or the receipt of pharmacotherapy. In this respect, the studies of
Kuzman et al. (2009) and Takahashi et al. (2010) warrant particular discussion.
Kuzman et al. (2009) analyzed gene expression in whole blood from 32 treatment-
naive patients presenting with their first psychotic episode suggestive of schizo-
phrenia compared to the same number of age- and gender-matched control cases.
Although a total of 180 probes were found to be differentially expressed between
the psychosis and nonpsychosis control group, these probes were not used in
conjunction with methods of class prediction to construct and evaluate the
predictive accuracy of a diagnostic gene expression classifier. Further, since
Kuzman et al. (2009) have not deposited their microarray gene expression data
in a public repository, it is not possible to assess the accuracy with which these
genes can distinguish psychosis cases from controls.
Takahashi et al. (2010) also analyzed gene expression in whole blood from 52
patients with schizophrenia or schizophreniform disorder recruited across six
outpatient clinics in Japan and 49 age-matched controls. Although 44 of these
patients were drug-naive or drug-free, eight patients were identified as antipsy-
chotics-naive, as they were taking antidepressants, benzodiazepines, or mood
stabilizers for prodromal symptoms. The samples were divided into a training
data set (35 schizophrenia and 33 controls) and an independent test data set (17
schizophrenia and 16 controls) for hold-out validation. ANNs and forward feature
THE UTILITY OF GENE EXPRESSION IN BLOOD CELLS 53
selection were used to identify 14 probes whose expression in whole blood could
discriminate schizophrenia cases from controls in the training data with 91.2%
accuracy as assessed by threefold cross-validation. The 14 probes in this diagnos-
tic gene expression classifier consisted of eight genes (CINP, DAOA, INSL3, LIPH,
MAP1D, NAF1, PGRMC1, and TDRD9) and six expressed sequence tags. This
diagnostic gene expression classifier was capable of correctly predicting 14/17
patients and 15/16 controls in the hold-out test data set resulting in a classification
accuracy of 87.9%. This pilot study clearly demonstrates the feasibility of using
blood gene expression to diagnosis schizophrenia; however, it still suffers from a
number of limitations. First, it does not appear that a globin reduction procedure
was employed when analyzing gene expression in whole blood. Second,
Takahashi et al. (2010) stated that gender was significantly different between
their schizophrenia and control groups in both the training (p ¼ 0.014) and the
test data sets (p ¼ 0.049) as measured by a chi-square test. Although attempts
were made to filter out gender-related genes, the authors stated that this study
should be repeated in a gender-matched cohort to ensure that the disease itself is
being diagnosed by the classifier (Takahashi et al., 2010). Third, this study could be
improved by the inclusion of schizophrenia-related neuropsychiatric disorders
(e.g., bipolar disorder) into the control group to confirm the specificity of the
classifier for schizophrenia. Finally, the diagnostic accuracy of this 14-probe
classifier should be validated in a cohort outside Japan. The study of Kuzman
et al. (2009), which analyzed schizophrenia patients in Croatia, would be well-
suited to the external validation of this Japanese study if the microarray data from
both studies were publicly available.
The maximization of sample size increases discriminatory and inferential
power, which is essential for any biomarker study of complex and heterogeneous
psychiatric disorders. As such, some groups have attempted to exploit the vast
stocks of blood-derived lymphoblastoid cell lines (LCLs) that have been estab-
lished from psychiatric patients over the past several decades as a potential source
of biomarkers. For several reasons, though, this approach is not entirely advisable.
Principally, this recommendation is based on scientific grounds, or more precisely,
the lack of scientific evidence supporting the suitability of LCLs for some types of
biomarker analysis. Clearly, blood cells immortalized by transformation with
Epstein-Barr virus produce faithful copies of DNA suitable for genomic analy-
sis—this is the premise on which such LCL repositories are founded—however,
there is less evidence that the epigenomic and transcriptomic patterns of LCLs
faithfully recapitulate profiles seen in fresh blood cells. Further, as LCLs are
necessarily far removed from the time of blood draw and the corresponding
clinical condition of the patient, LCL gene expression may have biomarker
potential only in reflecting biological ‘‘traits’’ and not ‘‘states.’’ On average,
31% of the variance in a gene’s expression level in LCLs is heritable (with
some being almost completely heritable and others entirely nonheritable;
54 CHRISTOPHER H. WOELK ET AL.
McRae et al., 2007). Thus, gene expression in LCLs may reflect, to some extent,
the effects of regulatory polymorphisms and can therefore be examined as a
partially heritable trait (but not state) in biomarker studies. Yet, Choy et al.
(2008) have shown that gene expression in LCLs exhibits significant variability
from day to day, as well as considerable susceptibility to nongenetic confounders,
such as baseline growth rates, the metabolic state in culture, transformation
efficiency, and biological noise (Choy et al., 2008). Studies of gene expression in
freshly drawn blood samples are certainly not immune to potential confounds, but
most of those can be controlled through careful study planning and ascertain-
ment, or detected and accounted for in statistical models.
An excellent and illuminating study by Min et al. (2010) was devised to
establish the degree of comparability of gene expression in freshly drawn and
transformed blood cell samples using multiple preparation protocols. Unfortu-
nately, the results indicated that gene expression signatures differed broadly
between each preparation, suggesting that the transcriptomic signature of LCLs
is not a suitable proxy for that derived from fresh blood cell samples. We
characterize this result as unfortunate, since we understand the desirability of
utilizing our precious existing resources to maximize the output of biomarker
discovery effort. Yet, the transcriptomic and regulatory disparities between fresh
and transformed blood samples may, in part, explain why Matigian et al. (2008)
found less than promising results in their study of schizophrenia, with no genes
showing even nominally significant dysregulation at a twofold change in the
patient group relative to a matched-control group. With an eye toward the future
and anticipating the desirability of immediate laboratory-based identification of
biomarkers in freshly drawn blood samples, coupled with the limitations of LCLs
identified above, we recommend that biomarker samples should be obtained
anew from patients at the time they are studied, and the precise clinical state of
the patient at that time should be noted. This practice will ensure that putative
biomarker discoveries may generalize more quickly to eventual clinical practice.
VI. Pharmacogenomics
in blood cells has already been used to predict treatment outcomes for glucocorti-
coid treatment of asthma (Hakonarson et al., 2005), interferon-a and ribivarin
treatment of hepatitis C virus (Lempicki et al., 2006; Tateno et al., 2007), angio-
genesis inhibitor SU5416 treatment of colorectal cancer (DePrimo et al., 2003),
interferon-b treatment of multiple sclerosis (Baranzini et al., 2005; van Baarsen
et al., 2008), and Torisel (CCI-779) treatment of renal cell carcinoma (RCC;
Burczynski et al., 2005). Specifically, Burczynski et al. (2005) identified 30 genes
in PBMCs whose expression could be used to predict good versus poor outcome
(i.e., time to progression) for RCC patients when subjected to Torisel treatment
with 74% accuracy as assessed by LOOCV in the training data, and 85%
accuracy in a test data set used for hold-out validation.
Neuropsychiatric disorders represent important targets for pharmacogenomic
studies. In schizophrenia, for example, 25–40% of patients fail to respond to their
first treatment option and 10–20% of those that do respond react with adverse
effects of clinical importance (Broich and Moller, 2008). Pharmacological studies
of neuropsychiatric disorders have primarily focused on associating single nucleo-
tide polymorphisms (SNPs) with treatment outcome. The majority of these studies
have used a candidate gene approach and are thus pharmacogenetic in scope
rather than whole genome-based pharmacogenomic analyses (Alenius et al., 2008;
Kato et al., 2008, 2009; Kwon et al., 2009; Need et al., 2009; Xu et al., 2010; Yasui-
Furukori et al., 2006). Recently, genome-wide association studies (GWAS) have
been used in true pharmacogenomic approaches to identify SNPs that could
predict responses in schizophrenia patients when treated with antipsychotics
(McClay et al., 2011) and in patients suffering from depression when treated
with antidepressants (Ising et al., 2009). In addition to these GWAS approaches,
the utility of gene expression in the blood for predicting treatment outcomes and
adverse effects in neuropsychiatric patients should now be determined.
when RNA is isolated from whole blood it is recommended that a globin reduc-
tion step be performed (Liu et al., 2006; Winn et al., 2010).
It is unlikely that the ultimate diagnostic classifier for a particular neuropsy-
chiatric disorder will rely solely on gene or miRNA expression data. The most
accurate classifier will probably incorporate a whole range of potentially diagnos-
tic criteria that may include any of the following: demographic variables, copy
number variation data, methylation patterns, SNPs, and protein expression. With
respect to the latter criteria, Schwarz et al. (2010) recently developed a diagnostic
protein expression classifier for schizophrenia with a classification accuracy of
77% for drug-naive patients. This study is fully reviewed in Chapters ‘‘The
application of multiplexed assay systems for molecular diagnostics’’ by Schwarz
et al. and ‘‘Algorithm development for diagnostic biomarker assays’’ by Izmailov
et al.
Genomics technologies are rapidly evolving. Soon, gene and miRNA expres-
sion analysis using microarrays will be replaced by next-generation sequencing
technologies. These technologies include Illumina’s HiSeq and Genome Analyzer
platforms, Applied Biosystems’ SOLiDTM system, and 454 Life Sciences’
Genome Sequencer FLX system, which are used to perform RNA-Seq and
miRNA-Seq for the assessment of gene and miRNA expression, respectively.
RNA-Seq analysis of gene expression has several advantages for constructing
diagnostic gene expression classifiers compared to microarray analysis including
a greater dynamic range, the sequencing of SNPs in coding regions, and the
identification of novel coding regions and alternatively spliced transcripts (Wang
et al., 2009). As the cost of sequencing continues to diminish and the software tools
for analyzing next-generation sequencing become more available (Oshlack et al.,
2010), the possibility of constructing more accurate diagnostic gene expression
classifiers for neuropsychiatric disorders will increase.
Acknowledgments
This work was performed with the support from ‘‘NARSAD: The Brain and
Behavior Research Fund,’’ in the form of a Young Investigator Award, and the
Sidney R. Baer, Jr. Prize (S.J.G.) and Lieber Prize Award (M.T.T.) for Schizophre-
nia Research. Additional support was derived from the Genomics Core at the
UCSD CFAR (AI36214), the San Diego Veterans Medical Research Foundation,
and other research grants from the National Institutes of Health (MH081755,
MH085240, AI087164). This chapter is based upon work supported in part by the
Department of Veterans Affairs (VA), Veterans Health Administration, Office of
Research and Development. The views expressed in this chapter are those of the
authors and do not necessarily reflect the position or policy of the Department of
58 CHRISTOPHER H. WOELK ET AL.
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PROTEOMIC TECHNOLOGIES FOR BIOMARKER STUDIES IN
PSYCHIATRY: ADVANCES AND NEEDS
Abstract
Abbreviations
I. Introduction
II. The Social Impact of Psychiatric Disorders
III. The Role of Proteomics in Psychiatry
A. What Has Been Done So Far?
IV. Proteomic Studies in Psychiatry: What Methods Have Been Used to Date?
A. Sample Preparation
B. Two-Dimensional Gel Electrophoresis and Mass Spectrometry
C. Shotgun Proteomics
D. SELDI-TOF
E. Metabolomics
F. Multiplex Analyte Profiling Approach
G. What Is the Best Method for Proteome Characterization?
V. Underexplored Proteomic Methods in Psychiatry Studies
A. Phosphoproteomics
B. SILAC
C. MALDI Imaging
VI. The Importance of Validation Experiments in Proteomics for Biomarker
Discovery in Psychiatry
A. Validation Technologies
VII. Clinical Translation
VIII. Summary
Acknowledgments
References
Abstract
In the case of psychiatric conditions, however, proteomic advances have had a less
profound impact. Here, we outline the necessity of improving and applying
proteomic methods for biomarker discovery and validation in the field of psychiat-
ric disorders. While proteomic-based applications in neurosciences have increased
in accuracy and sensitivity over the past 10 years, the development of orthogonal
validation technologies has fallen behind. These issues are discussed along with the
importance of integrating systems biology approaches and combining proteomics
with other research approaches. The future development of such technologies may
put proteomics closer to clinical applications in psychiatry.
ABBREVIATIONS
I. Introduction
launched in 1999 by Andrew Link, increasing the capacity for proteome charac-
terization (Link et al., 1999). Proteomics in psychiatric studies followed the ten-
dency of delivering accurate and sensitive technologies for biomarker discovery
but did not pay sufficient attention to validation technologies. Although the
usefulness of Western blot (WB) and immunoassay methods are recognized,
newer high-throughput and more sensitive tools are needed. Recently, a valida-
tion method termed selective reaction monitoring (SRM) has emerged which
shows more promise in this area. Besides the requirement of new validation
technologies, new systems biology approaches of merging proteomics with tran-
scriptomic and metabolomic methods are also needed. Here, we present and
discuss current proteomics technologies and the future steps that will allow this
relatively new scientific methodology to advance the identification, validation,
and clinical deployment of biomarkers in the challenging field of psychiatric
disorders.
The proteome is defined as ‘‘the total set of expressed proteins by a cell, tissue
or organism at a given time under a determined condition’’ (Wilkins et al., 1996).
Proteomics is the study of proteomes and may also include the study of posttrans-
lational modifications and protein–protein interactions. In 1999, the first proteo-
mics study of cancer was published (Banks et al., 1999). As of March 2011, a
PubMed search for ‘‘cancer and proteomics/proteome’’ returned approximately
4201 articles, showing the large investment into proteomic analyses of these
conditions. Proteomic studies of cancer have thus far led to breakthroughs in
diagnosis and treatment (see Chapter ‘‘Challenges of introducing new biomarker
products for neuropsychiatric disorders into the market’’ by Bahn et al.), support-
ing the importance of the technology as a tool for the comprehension of biochem-
ical pathways, biomarker discovery, and identification of therapeutic alternatives
for diseases. The first proteomics paper in psychiatric disease was published
in 2000 (Johnston-Wilson et al., 2000) and a current PubMed search for the
appropriate terms returns only 144 relevant articles.
These comparisons have led us to the conclusion that new applications are
necessary in psychiatric studies. Biomarker studies on psychiatric disorders pres-
ent peculiar hurdles such as the inherent difficulties in accessing relevant
biological materials, since the main manifestations appear to be in the brain.
Moreover, the heterogeneity of brain tissue may be challenging considering, for
example, that decreases in synaptic proteins could result from cell-specific
mechanisms or a functional change such as lack of input to a particular brain
region. As a consequence, the identification of correlating biomarkers in the
periphery is required before these can be clinically useful.
Unbiased approach
Candidate Qualification and
Discovery phase
discovery quantification
Biological validation
(On a larger sets of samples)
Assay development
(e.g. immunoossays/SRM)
Biased approach
Validation phase
Commercialization
IV. Proteomic Studies in Psychiatry: What Methods Have Been Used to Date?
A. SAMPLE PREPARATION
Control Disease
Molecular weight
Isoelectric point
Image analyses
Spots extraction
In gel digestion
INTENSITY
Mass spectrometry
M/Z
C. SHOTGUN PROTEOMICS
1. Label-Free MS
The theoretical assumption that the chromatographic peak area of a given
peptide should correspond to its concentration (Chelius and Bondarenko, 2002)
was experimentally proven through studies of peptide ion current intensities
(Chelius and Bondarenko, 2002; Levin et al., 2007). This is the main principle of
label-free quantitative proteome analysis which seems to be the easiest way of
comparing proteomes, since no labeling for proteome quantification is required
and, in contrast to other proteomic profiling techniques, there are no limits
regarding number of samples for analysis, which makes this approach well-suited
for clinical studies. This is important as analysis of larger samples cohorts provides
greater statistical power, which is essential in biomarker discovery projects to
minimize false-positive results.
Label-free proteomics can be performed basically using four distinct
approaches. Two of these, ‘‘spectral counting’’ and ‘‘data-dependent acquisition
(DDA)-based ion counting,’’ are data dependent, whereas the other two, ‘‘MS
survey scan-based ion counting’’ and ‘‘data-independent analysis (MSE),’’ are
acquired in an MS survey scan (Levin and Bahn, 2010). Considering that in
MSE (Li et al., 2009), intact peptides and fragmented peptides are intermittently
measured in a single experiment at high sampling rate, this seems to be the most
76 DANIEL MARTINS-DE-SOUZA ET AL.
Light
RELATIVE ABUNDANCE
430.5
871.5
Heavy
658.3
935.6
The calculated ratios of different reporter tag intensities will provide relative
quantitation for the peptide/protein in question. The larger number of mass tags
also provides greater flexibility in experiments. For example, this will allow direct
comparison of samples from the same subject in longitudinal studies of drug effects.
The ICPL and iTRAQ methodologies have been used successfully in recent
proteome analyses of schizophrenia brain tissue (Martins-de-Souza et al., 2009a,b,
2010d), providing evidence of proteins and biological pathways which are altered
in the disease.
In general, stable isotope labeling methods can be used at the protein or
peptide level and can be combined with most proteomic separation approaches,
providing accurate quantification. The disadvantages of stable isotope labeling,
however, is that the targeted proteins or peptides are labeled after sample prepa-
ration procedures such as protein extraction, subproteome isolation, or even
generation of peptides, which can increase the probability of introduced experi-
mental errors, resulting in decreased accuracy. Moreover, the maximum number
of eight isotope tags is a limiting factor for most clinical applications, although
different time points of the same patient can be measured in one experiment.
3. In Vivo Labeling
In vivo labeling methods avoid some of the drawbacks associated with the
stable isotope labeling approach (Filiou et al., 2011). Although these methods are
not readily suitable for studies on living human tissues, they can be used easily in
cell culture and animal model experiments. In this approach, stable isotopes such
as 2H, 13C, 15N, or 18O are introduced in vivo such that they can be incorporated
into newly synthesized proteins. Since these are natural isotopes, there should
theoretically be no effects on the biological systems into which they are
incorporated. In comparative proteomic studies using this method, labeled and
unlabeled versions of the proteome extracts can be combined prior to any sample
preparation avoiding the types of experimental errors that are seen with the
postlabeling approaches (Gouw et al., 2010).
In psychiatric studies, 15N metabolic labeling has been applied to proteomic
studies of a mouse model of trait anxiety (Kromer et al., 2005). High, normal, or
low anxiety-related behavior (denominated HAB, NAB, and LAB, respectively)
mice were fed with 15N-labeled diets from cultured algae or bacteria resulting in
up to 92% of 15N incorporation in plasma and brain tissue (Frank et al., 2009). No
effects on development, organ morphology, physiology, or reproductive ability
due to the introduction of isotopes into the feed were detected (Wu et al., 2004;
Frank et al., 2009). However, alterations in behavioral phenotype and in compo-
nents of the proteome were observed (Frank et al., 2009; Filiou et al., 2011).
PROTEOMIC TECHNOLOGIES FOR BIOMARKER STUDIES IN PSYCHIATRY 79
D. SELDI-TOF
E. METABOLOMICS
All of the molecular profiling methods described here have their strengths and
weaknesses, which have been described. Therefore, we recommend the combined
use of different methodologies as this would not only maximize proteome cover-
age, but it would also result in more comprehensive information on the relevant
molecular pathways involved in the disease process and give increased confidence
in the biomarker candidates identified.
PROTEOMIC TECHNOLOGIES FOR BIOMARKER STUDIES IN PSYCHIATRY 81
A. PHOSPHOPROTEOMICS
B. SILAC
Stable isotope labeling by/with amino acids in cell culture (SILAC) is an in vivo
labeling method, which has been used in conjunction with proteomic
studies. 7SILAC consists of growing the cells of interest in medium containing
specific 13C- or 15N-labeled amino acids, such as lysine or arginine, for quantita-
tive comparison of proteins in cells which have been cultured in unlabeled media
(Ong et al., 2002). Proteome quantification relies in the same principal as stable
isotope labeling as described above.
SILAC could be used to provide precise quantitative proteomic information
in cell culture models of psychiatric disorders as described previously (Steiner et al.,
2010; Martins-de-Souza et al., 2011a,b). Primary cortical neurons from a mouse
model of fragile X syndrome (FXS) and wild-type mice were cultured using the
SILAC approach in order to identify changes in synaptic proteins (Liao et al.,
2008). Interestingly, the SILAC concept can also be applied to animal models by
incorporation of 13C-labeled lysine in the chow for subsequent quantitative
comparisons (Krüger et al., 2008).
82 DANIEL MARTINS-DE-SOUZA ET AL.
C. MALDI IMAGING
2500
2000
$ Millions
1500
1000
500
0
2008 2009 2010 2015
FIG. 4. Evolution of central nervous system biomarker marker (BBC Research, report: BIO074A,
October 2010).
A. VALIDATION TECHNOLOGIES
peptides of interest are selected, (3) selected peptides are fragmented and the
fragments (SRM transitions) are aligned, (4) SRM transitions are singly detected,
and (5) data of individual SRM transitions are combined in order to provide a final
intensity of the previous selected peptide, which consequently will provide quantita-
tive information. The schematic representation of SRM analyses is represented in
Fig. 5. The quantitative power of SRM relies on the fact that a single peptide is
accurately quantified several times, since each peptide is broken into multiple
fragments (Fig. 5, Q2). Each SRM transition (or peptide fragment) will be quantified
and, together with data from the other fragments, provide accurate quantitative data
for a given peptide. The combined quantitative data from different peptides from the
same protein can then be used to derive the absolute and relative quantitation of that
protein. It is important to highlight that the sensitivity of this method may reach the
attomole range. Moreover, SRM allows precise characterization of several peptides
and posttranslational modifications such as phosphorylation, acetylated lysine resi-
dues, ubiquitination, and glycosylation.
SRM experiments have been employed in proteomic studies (Armenta et al.,
2010; Zhang et al., 2011) but have thus far not been applied in studies of
psychiatric disorders. The high-throughput nature and multiplexing capability
of SRM (it is possible to measure peptides from several proteins simultaneously)
indicate that it could be useful as a potential technique in validation analyses of
samples from large human cohorts. This technology is also of interest as it can be
used in both preclinical and clinical trials, in which there is increased usage of
biomarkers as surrogate endpoints. Moreover, the multiplexing capability makes
SRM a useful methodology for use in clinical pipelines for detection of diagnostic,
prognostic, and treatment-related biomarkers.
2. ELISA
ELISA is one of the main techniques used for detection and quantification
of protein expression with high sensitivity and good reproducibility, which allows
the analysis of protein samples in a microplate format. This technique requires the
Q1 Q2 Q3
Intensity
Time
Peptide
Peptide Fragment
fragmentation
selection accurate
and fragment
measurement
selection
availability of two antibodies for each target protein. One antibody is used for the
capture of the protein of interest and the other is used for detection. This technology
can often be used easily off-the-shelf although it sometimes requires optimization
steps when there are large numbers of variables including differences in antibody
specificity, and reagent compatibility with conditions such as buffer components in
the protein extracts or media of interest. This technique has already been used
extensively in psychiatric and neurological disorders including the measurement
of b-amyloid peptides in Alzheimer’s disease (see review Humpel, 2010), profiling
of insulin-related molecules in schizophrenia (Guest et al., 2010), and for validation of
biomarker candidates identified by LC–MSE profiling of serum from schizophrenia
patients (Craddock et al., 2008; Levin et al., 2010).
3. Western Blot
WB is another immunological method for protein detection and quantitation
that it is often used in preference to ELISA in studies of tissue extracts. The method is
only deemed semiquantitative although it has an advantage over ELISA in that WB
affords visualization of protein bands, affording confidence in specificity of the
antibodies used. This also has the added advantage that posttranslational changes
such as proteolysis can be visualized by a change in apparent MWor pI, for example.
Other advantages include the possibility of using one secondary antibody in the
detection of several primary antibodies and the availability of secondary antibodies
offering different types of detection. One main disadvantage of WB is that the
secondary antibody may bind nonspecifically to other proteins, particularly immu-
noglobulin chains, in the extract. A potentially powerful application would involve
combination of ELISA and WB methods as shown in a recent study (Carlino et al.,
2010). The ELISA method gave a result indicating a decrease in serum brain-
derived neurotrophic factor (BDNF) in schizophrenia compared to control patients,
and WB showed that this was associated with an increase in pro-BDNF and mature
BDNF, with a concomitant decrease in a truncated form of the molecule. This
combined analysis allowed the authors to correlate the expression of different forms
of serum BDNF with cognitive performance.
4. Functional Genomics
Gene expression mediates cellular activity and first gives rise to synthesis of
messenger RNA. In neuroscience and especially in neurological disorders, micro-
arrays constitute the most common technology used, in association with qPCR
validation. One example of multiplatform analysis is the characterization of QKI
gene expression in suicide victims who suffered from major depressive disorder
(Klempan et al., 2009). The authors identified a reduction of QKI mRNA levels in
cortical, hippocampic, and amygdala regions of suicide victims compared to
control subjects. The microarray findings were confirmed by qPCR and also by
reduced expression of the encoded proteins, as shown by WB analysis. There are
86 DANIEL MARTINS-DE-SOUZA ET AL.
5. Tissue Microarray
Despite the existence of many well defined molecular technologies such as
ELISA, PCR, and WB, one main shared disadvantage of these methods is that
they are subject to artifactual effects incurred by the tissue or body fluid before
analysis. Moreover, proteins or genes might have differential expression regarding
their subregional distribution and therefore identifying their specific localization is
crucial. Tissue microarray (TMA) is a useful technique for providing information
on intracellular distribution of molecules. This method consists of hundreds of
tissue cores assembled as an array which allows the simultaneous analysis of
multiple samples and biomarkers in a single study. Most of the assays used in
TMA are immunohistochemical in nature but can also include in situ hybridiza-
tion methods, mostly in cases involving tumor biopsy, and also in neurodegenera-
tive and inflammatory diseases.
6. Protein Arrays
Protein arrays typically comprise multiple proteins or antibodies arrayed in
separate locations on a microtiter plate or other surface, to allow simultaneous
analysis of multiple protein targets. Such arrays provide a good platform for
efficient profiling of protein expression (Table I) and also for identification of
interactions between other proteins, antibodies, drugs, or ligands. Given that this
is a multiplex format, other advantages include the need for lower samples volume
and antibodies and, therefore, the high reproducibility and associated low costs.
Table I
FEATURES OF TECHNOLOGIES CURRENTLY USED FOR BIOMARKER VALIDATION.
Biomarkers validation is important in all fields, but the optimal analytic plat-
form which allows their translation into the clinic is crucial. Findings at the
proteomic level must be developed as user friendly and robust assays for ease of
use in the clinic. Only in this way can biomarkers realize their full potential leading
to improved diagnostic accuracy, better disease classification and, ultimately, to
personalized medicine strategies for more effective treatment affected populations.
To select the best candidate for clinical evaluation, optimization of the pharmaco-
kinetic parameters is required (Sarker and Workman, 2007; Sarker et al., 2007).
The use of validated biomarkers in clinical trials remains low. This could be
important as only 5% of oncology drugs which have entered the clinical trial phase
have been commercialized. Technologies and biomarkers are available for clinical
trial use in areas such as cancer, including microRNAs (Bartels and Tsongalis, 2009;
Scott et al., 2007; Volinia et al., 2006), SRM standards (Chen et al., 2010), and gene
transcripts (Rubinstein et al., 2010). However, only few have been used in the area of
psychiatric disorders. Recently, we launched a blood test for schizophrenia termed
VeriPsychTM, as described above. This test will help clinicians to confirm diagnosis
of this pathology and give opportunities for the investigation and development of
future treatment for this devastating disorder which affects more than 20 million
people worldwide (World Health Organization). This represents a successful model
of translational medicine applied in psychiatry, which bridged proteomic findings to
result in a marketed product that will improve patients’ lives.
VIII. Summary
Acknowledgments
This research was supported by the Stanley Medical Research Institute (SMRI)
and the European Union FP7 SchizDX research programme (grant reference
223427).
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CONVERGING EVIDENCE OF BLOOD-BASED BIOMARKERS FOR
SCHIZOPHRENIA: AN UPDATE
Abstract
I. Introduction
II. Methodology
A. Compilation of Literature Serum/Plasma Biomarkers
B. Compilation of In-House Serum Biomarkers
C. In Silico Functional Pathway Analysis
III. Results
A. Evidence from the Literature Review
B. Evidence from In-House Studies: Biomarkers of First-Onset Schizophrenia
C. Functional and Pathway Analysis: Overall Evidence
IV. Discussion
A. Genetic, Epidemiological, and Animal Model Studies
B. Evidence for Innate and Adaptive Immune Response Activation in Schizophrenia
C. Innate Immune Response: APR Signaling and Hepatic Metabolism
D. Adaptive Immune Response: Type-1 and Type-2 Response Imbalance
E. Effects of Antipsychotic Drugs on Immune-Related Processes
F. Glucocorticoid Receptor Signaling
G. Type-1 and Type-2 Immune System Rebalance
H. Evidence for Type 1/Type 2 Immune Response Imbalance in the CNS
V. Conclusion and Perspectives
Acknowledgments
References
Abstract
This chapter has carried out a review of the literature and combined this with
the results of in-house studies to identify candidate blood-based biomarkers for
schizophrenia and antipsychotic drug response. Literature searches retrieved 185
publications describing a total of 273 schizophrenia biomarkers identified in
I. Introduction
Diagnosis currently relies on subjective assessments obtained from the patient’s self-
reported symptoms, mental status examinations, and the clinician’s observations in
line with the classifications listed in the Diagnostic and Statistical Manual of Mental
Disorders 4th Edition (DSM-IV) or the International Statistical Classification of
Diseases and Related Health Problems 10th Revision (ICD-10). Correct diagnosis
may take months to years to complete and there is considerable room for error as
symptoms may overlap with those in a number of psychiatric conditions (e.g., drug-
induced psychoses, delirium, and dementia), psychotic mood disorders, and person-
ality disorders (Lakhan and Kramer, 2009; Lakhan et al., 2010). As a result, there has
been a recent and rapid shift toward the study of specific and sensitive molecular
biomarkers in psychiatry. The hope is that such biomarkers will complement the
purely syndromal diagnostic procedures. Biomarker research has already had a
positive impact in other branches of medicine including oncology, rheumatology,
cardiology, and obstetrics (Cook, 2008). Recently, Psynova Neurotech successfully
launched the first biomarker-based blood test designed to aid psychiatrists in the
diagnosis of recent-onset schizophrenia (http://www.psynova-neurotech.com/
default.htm; Schwarz et al., 2010; see Chapter ‘‘The application of multiplexed
assay systems for molecular diagnostics’’ by Schwarz et al.).
Relative to organ tissue (e.g., brain), body fluids such as blood, urine, and
cerebral spinal fluid (CSF) represent more easily accessible sources for detection of
systemic biomarkers. The blood, for example, can be sampled using standardized
and routine clinical procedures without significant patient discomfort. The main
advantages associated with using blood as a source of biomarkers include the fact
that it is possible to design standardized sample collection procedures, it is
available in sufficient quantities, and it can be sampled on multiple occasions
with relative ease (Lakhan and Kramer, 2009). Biomarker studies in psychiatry
have undergone a fundamental methodological shift from searching for cause to
estimating the probability that a condition is present or may develop (Singh and
Rose, 2009). Diagnostic biomarkers may be used for identification of the disease
early in its course. This may be critical for remission since evidence suggests that
delays in diagnosis and intervention lead to poorer prognoses (Lakhan and
Kramer, 2009). Biomarkers with prognostic value may facilitate prediction
of disease development, the likely course and outcome of illness, and certain
disease-associated behaviors and personality traits. Further, biomarkers may
also enable prediction of the type, timing, course, and response to treatment,
and may ultimately enable disease subtyping and patient stratification. Integra-
tion of drug-response (DR) biomarkers into drug discovery programs may pro-
mote development of novel therapeutics along with a personalized medicine
approach. Biomarkers may inform whether such therapeutics are worth pursuing
and facilitate subsequent go/no go decisions regarding safety and efficacy.
This information could be applied to drug design to facilitate selective targeting
of relevant patient populations and aid dose selection/adjustment which will
ultimately enable selection of potential drug responders (Singh and Rose, 2009;
Tesch et al., 2010).
98 MAN K. CHAN ET AL.
II. Methodology
The keywords used for Pubmed searching included ‘‘schizophrenia’’ and ‘‘blood’’
or ‘‘serum’’ or ‘‘plasma’’ or ‘‘CSF’’ or ‘‘cells.’’ Only schizophrenia studies reporting
protein or mRNA biomarkers with gene names were included. Only articles written
in English and published between 1995 and April 2010 were included. Comprehen-
sive details from these studies were recorded in an excel spreadsheet and the informa-
tion compiled from each article included biomarker name (gene and protein names),
type [diagnostic (D), DR, and diagnostic or drug-response (DorDR) biomarkers],
assay type [e.g., immunoassay, Western blot, MS, reverse transcription-polymerase
chain reaction (RT-PCR), etc.], study finding (increase or decrease in level), study
design (number of patients/controls), sample source (e.g., serum, CSF, plasma, etc.),
Pubmed ID (PMid), comments on each article (e.g., controls for confounding factors),
journal title, and impact factor. The list of significant biomarkers extracted was
subjected to in silico functional pathway analysis as described below.
CONVERGING EVIDENCE OF BLOOD-BASED BIOMARKERS FOR SCHIZOPHRENIA 99
The lists of molecules compiled from the literature and in-house datasets were
subjected to functional pathway analysis using the Web-based Ingenuity Pathways
Analysis (IPA) tool (IngenuityÒ Systems; www.ingenuity.com). Briefly, this analysis
was used to identify the most significant biological functions (‘‘diseases and
disorders,’’ ‘‘molecular and cellular functions,’’ and ‘‘physiological system devel-
opment and function’’) and canonical pathways associated with the molecules.
For functional analysis, right-tailed Fisher’s exact test was used to calculate a
100 MAN K. CHAN ET AL.
Table I
DEMOGRAPHIC DETAILS OF SUBJECTS THAT PARTICIPATED IN THE IN-HOUSE STUDIES (MEAN SD).
p-value determining the probability that the assignment of each biological func-
tion and/or disease was due to chance alone. For the canonical pathway analysis,
the significance of the association between molecules in the dataset and the
canonical pathway was measured in two ways: (1) the ratio of the number of
molecules from the dataset associated with the pathway was divided by the total
number of molecules known to map onto that pathway; (2) Fisher’s exact test was
used to calculate a p-value, determining the probability that the association
between the biomarkers in the dataset and the canonical pathway was explained
by chance alone.
III. Results
Protein name Gene name Diagnostic Drug response Diagnostic or drug response Total
(Continued)
Table II (Continued)
Protein name Gene name Diagnostic Drug response Diagnostic or drug response Total
Diagnostic or D
(15) markers (40)
HP IGHM
APOA4 MDK
Literature AHSG TF
B2M APOA2
CFB APOC1
DEFA1 CD5L
IL12 F13B Diag-prog or DorDR
TTR markers (76)
(5) (5)
SCGB1A1 APOA1
APOD ALB
IGF1 IFNG
(15)
IL6R SERPINA1
RAGE ICAM1 CRP INS IL4 A2M IL16 APOH
IL1B NGF BDNF APCS IL18 CAT
IL8 TNF IL6 C3 IL1A CP
IL1RN IL2 S100B CCL22 IL3 FGA
CCL4 KITLG GPX
IL2RA LEP PRL CCL5 LTA GSTA1
FGF2 CEACAM5 MMP9 IGFBP1
GHRL CSF2 SELL MMP2
CCL11 CXCL5 SERPINE1 MUC16
INHBB
ADIPOQ DPP4 SST NCAM
FSHB
IL2R EDN1 TGFB1 RELN
OXT F3 TIMP1 SERPINA7
SOD1
F7 TNFRSF14 THPO
(5) (4) GH1 TNFRSF1B CCL2
IL12A VCAM1 IL10
IL12B VEGFA TNFRSF1A
IL13 ACPP VWF
IL15 APOC3 EGF
Drug-response or (54)
DR markers (29)
common among all three biomarker categories, 5 were grouped into the D and
DR groups, 5 were common between the D and DorDR groups, and 4 were
common between both the DR and DorDR groups (Fig. 1).
(Continued)
Table III (Continued)
(Continued)
Table III (Continued)
1. Biological Functions
‘‘Immunological disease,’’ ‘‘inflammatory response,’’ and ‘‘respiratory dis-
ease’’ were the most significant diseases and disorders associated with the literature
and with the MAP data (Fig. 2). Not surprisingly, the LC–MS studies did not share
the top diseases and disorders in common with the literature or MAP data as this
method typically analyzes the medium-high abundance portion of the proteome
(Higgs et al., 2008). The proteins identified by the LC–MS studies were predomi-
nantly associated with the ‘‘cardiovascular’’ and ‘‘metabolic component’’ in
schizophrenia, consistent with some of the current hypotheses on this disorder
(Newcomer, 2007). This finding also highlighted the usefulness of unbiased MS
profiling for identification of novel disease-associated functions. The most signifi-
cant molecular and cellular functions in common between the literature and MAP
studies were ‘‘cellular movement’’ and ‘‘cell death,’’ and the most significant
molecular and cellular functions in common between the literature and the LC–
MS analysis were ‘‘lipid and molecular transport’’ and ‘‘small molecular biochem-
istry’’ (Fig. 2). ‘‘Antigen presentation,’’ ‘‘carbohydrate metabolism,’’ and ‘‘cell
morphology’’ were significantly associated with the MAP and LC–MS studies,
respectively. ‘‘Hematological system development and function’’ was among the
most significant physiological system development and functions associated with
the literature, MAP, and LC–MS studies. While ‘‘immune cell trafficking’’ and
‘‘tissue morphology’’ were shared between the literature and MAP studies, the
LC–MS studies had no other physiological system functions in common (Fig. 2).
2. Canonical Pathways
Canonical pathway analysis of the ‘‘diagnostic biomarkers’’ from the litera-
ture, MAP, and LC–MS studies provided further evidence for altered immuno-
logical and/or inflammatory signaling in schizophrenia (Fig. 2). ‘‘Acute phase
response (APR) signaling’’ was the top canonical pathway shared between all
the studies (Fig. 3A). The APPs involved in APR signaling included 10 positive
APPs [IL1RN, HP (haptoglobin), CRP (C-reactive protein), C3 (complement
112 MAN K. CHAN ET AL.
Table IV
SUMMARY OF LC–MS FINDINGS FROM THE TWO PATIENT COHORTS COMBINED.
Alpha-2-HS-glycoprotein AHSG # –
Apolipoprotein A-I APOA1 # –
Apolipoprotein A-II APOA2 # –
Apolipoprotein A-IV APOA4 # –
Apolipoprotein C-I APOC1 # –
Apolipoprotein D APOD # –
CD5 molecule-like CD5L # –
Immunoglobulin heavy constant mu IGHM # –
Transferrin TF # –
Coagulation factor XIII F13B # #
Ubiquitin-specific peptidase 54 USP54 – #
Creatine kinase CKMT2 – #
Growth regulation by estrogen in breast cancer 1 GREB1 – #
Putative N-acetylated-alpha-linked acidic dipeptidase FOLH1B – #
Serine/threonine kinase 38 like STK38L – #
Vang-like 1 VANGL1 – #
Probable ATP-dependent DNA helicase HFM1 HFM1 – #
Fibulin 1 FBLN1 – #
Polymerase (RNA) III (DNA directed) polypeptide A, POLR3A – #
155 kDa
Myoferlin MYOF – #
Actinin, alpha 2 ACTN2 – #
Peroxidasin homolog (Drosophila) PXDN – #
Leucine rich repeat containing 16A LRRC16A – #
Lumican LUM – #
Retinol-binding protein 4, plasma RBP4 – #
Gelsolin GSN – #
Serpin peptidase inhibitor, cladeF SERPINF2 – "
Synemin, intermediate filament protein SYNM – "
Neurexin 1 NRXN1 – "
Apolipoprotein L, 1 APOL1 – "
CDK5 regulatory subunit associated protein 2 CDK5RAP2 – "
Polymerase (DNA directed), theta POLQ – "
Collagen, type V, alpha 1 COL5A1 – "
Spermatogenesis-associated protein 21 SPATA21 – "
Microtubule-associated protein 2 MAP2 – "
RANBP2-like and GRIP domain containing 1 RGPD1 – "
Haptoglobin HP – "
Solute carrier family 25 SLC25A10 – "
Peroxisome proliferator-activated receptor gamma PPRC1 – "
PDX1C-terminal inhibiting factor 1 PCIF1 – "
Table V
TOP BIOLOGICAL FUNCTIONS.
Diagnostic Prognostic
First onset or chronic (literature) First onset (RBM) First onset First onset or chronic (literature)
(LC–MS)
Diseases and Inflammatory disease Inflammatory response Cardiovascular disease Inflammatory response
disorders (1.01 10 20–6.11 10 9, (1.85 10 46–9.17 10 12, (1.31 10 4–4.96 10 2, (4.91 10 18–5.22 10 8,
n ¼ 28) n ¼ 71) n ¼ 4) n ¼ 22)
Immunological disease Immunological disease Genetic disorder (1.34 10 4– Immunological disease
(2.25 10 20–6.18 10 9, (2.19 10 38–6.59 10 12, 4.96 10 2, n ¼ 21) (1.77 10 17–6.09 10 8,
n ¼ 26) n ¼ 59) n ¼ 19)
Inflammatory response Respiratory disease Neurological disease Genetic disorder (2.16 10 17–
(5.43 10 20–6.18 10 9, (9.16 10 33–8.11 10 12, (1.34 10 4–4.23 10 2, 4.68 10 8, n ¼ 23)
n ¼ 30) n ¼ 45) n ¼ 19)
Respiratory disease Hematological disease Psychological disorders Nutritional disease
(6.32 10 19–1.70 10 9, (9.16 10 33–8.11 10 12, (1.34 10 4–1.34 10 4, (4.16 10 17–1.17 10 9,
n ¼ 22) n ¼ 50) n ¼ 8) n ¼ 17)
Organismal injury and Renal and urological disease Metabolic disease (4.08 10 4–Endocrine system disorders
abnormalities (2.46 10 18– (1.45 10 28–6.52 10 16, 4.96 10 2, n ¼ 5) (1.24 10 16–4.58 10 8,
3.40 10 9, n ¼ 23) n ¼ 37) n ¼ 14)
Molecular Cellular movement Cellular movement Lipid metabolism (4.23 10 6– Cellular movement
and cellular (4.47 10 24–6.07 10 8, (2.10 10 46–9.17 10 12, 4.96 10 2, n ¼ 11) (8.14 10 23–6.46 10 8,
functions n ¼ 28) n ¼ 71) n ¼ 23)
Lipid metabolism (2.87 10 21– Cell-to-cell signaling and Small molecule biochemistry Cell-to-cell signaling and
7.13 10 9, n ¼ 25) interaction (1.70 10 45– (4.23 10 6–4.96 10 2, interaction (4.14 10 21–
8.56 10 12, n ¼ 74) n ¼ 15) 7.42 10 8, n ¼ 23)
Molecular transport Cellular growth and proliferation Molecular transport Cell death (5.82 10 19–
(2.87 10 21–6.82 10 9, (3.16 10 40–7.61 10 12, (4.78 10 6–4.97 10 2, 7.27 10 8, n ¼ 21)
n ¼ 26) n ¼ 75) n ¼ 12)
(Continued)
Table V (Continued)
Diagnostic Prognostic
First onset or chronic (literature) First onset (RBM) First onset First onset or chronic (literature)
(LC–MS)
Small molecule biochemistry Antigen presentation Carbohydrate metabolism Cellular growth and proliferation
(2.87 10 21–7.13 10 9, (1.83 10 35–4.15 10 12, (3.33 10 5–4.96 10 2, (2.29 10 18–6.16 10 8,
n ¼ 27) n ¼ 47) n ¼ 7) n ¼ 25)
Cell death (8.40 10 19– Cell death (5.46 10 33– Cell morphology (3.75 10 5– Lipid metabolism (7.28 10 18–
6.82 10 9, n ¼ 29) 4.78 10 12, n ¼ 71) 4.71 10 2, n ¼ 8) 7.26 10 8, n ¼ 20)
Physiological Hematological system Hematological system Organismal functions Hematological system
system development and function development and function (2.36 10 3–7.60 10 3, development and function
development (5.54 10 22–6.18 10 9, (2.52 10 43–9.17 10 12, n ¼ 3) (3.05 10 15–6.46 10 8,
and function n ¼ 31) n ¼ 73) n ¼ 21)
Immune cell trafficking Immune cell trafficking Organ morphology Immune cell trafficking
(5.54 10 22–6.07 10 9, (2.52 10 43–9.17 10 12, (2.44 10 3–1.77 10 2, (3.05 10 15–6.46 10 8,
n ¼ 29) n ¼ 66) n ¼ 6) n ¼ 19)
Tissue morphology Tissue development Embryonic development Tissue morphology
(3.35 10 17–6.18 10 9, (2.28 10 39–1.05 10 11, (2.54 10 3–1.88 10 2, (3.19 10 15–7.26 10 8,
n ¼ 26) n ¼ 60) n ¼ 4) n ¼ 21)
Organismal survival Tissue morphology Endocrine system development Behavior (1.15 10 13–
(3.43 10 17–2.78 10 9, (3.43 10 31–1.05 10 11, and function (2.54 10 3– 1.73 10 8, n ¼ 15)
n ¼ 22) n ¼ 57) 2.51 10 2, n ¼ 3)
Lymphoid tissue structure and Cell-mediated immune response Hematological system Digestive system development and
development (7.63 10 16– (5.73 10 27–9.17 10 12, development and function function (1.28 10 13–
1.17 10 9, n ¼ 16) n ¼ 34) (2.54 10 3–4.23 10 2, 1.28 10 13, n ¼ 7)
n ¼ 5)
CONVERGING EVIDENCE OF BLOOD-BASED BIOMARKERS FOR SCHIZOPHRENIA 115
Table VI
TOP CANONICAL PATHWAYS ASSOCIATED WITH DIAGNOSTIC OR DRUG-RESPONSE BIOMARKERS IDENTIFIED
FROM THE LITERATURE OR IN-HOUSE STUDIES (MAP OR LC–MS STUDIES).
Mixed disease stages First onset (MAP) First onset Mixed disease stages
(literature) (LC–MS) (literature)
component 3), FGA (fibrinogen alpha chain), AGT (angiotensinogen), CFB (com-
plement factor B), CP (ceruloplasmin or ferroxidase), FTH1 (ferritin), and PROS1
(proteins alpha)], 5 negative APPs [AHSG (alpha-2-HS-glycoprotein), TF (transfer-
rin), ALB (albumin), TTR (transthyretin), and IGF1 (insulin-like growth factor1)],
and 21 other APPs [IL6, TNF, IL1B (interleukin 1 beta), IL1A (interleukin 1 alpha),
IL8, MIF (macrophage migration inhibitory factor), IFN-g (interferon gamma),
APOA1 (apolipoprotein A-I), APOH (apolipoprotein H), VWF (von Willebrand
factor), SERPINA1 (serpin peptidase inhibitor cladeA), A2M (alpha-2-macroglob-
ulin), APCS (amyloid P component), APOA2 (apolipoprotein A-II), IL18 (interleu-
kin 18), IL6R (interleukin 6 receptor), SERPINE1 (serpin peptidase inhibitor
cladeE), TNFRSF1A (tumor necrosis factor receptor superfamily member 1A),
RBP4 (retinol-binding protein 4), SERPINF2 (serpin peptidase inhibitor cladeF),
and TNFRSF1B (tumor necrosis factor receptor superfamily member 1B)].
116 MAN K. CHAN ET AL.
Inflammatory disease
Organismal injury and
abnormalities
First-onset
First-onset RBM LC–MS
Diagnostic Immunological disease Diagnostic First-onset RBM Small molecule First-onset LC–MS
Inflammatory response Diagnostic Biochemistry Diagnostic
Respiratory disease Cellular movement Molecular transport
Cell death Lipid metabolism
Cardiovascular disease
Hematological disease Genetic disorder
Neurological Disease Carbohydrate metabolism
Renal and urological disease Antigen presentation Cell morphology
Psychological Disorders
Metabolic Disease
First-onset LC–MS
First-onset RBM
First-onset LC–MS Diagnostic
First-onset RBM Diagnostic
diagnostic Diagnostic
Immune cell trafficking
Tissue morphology Role of cytokines in mediating
Communication between immune cells
Communication between innate and
Hematological system adaptive immune cells
Hepatic fibrosis Acute
development and
phase
function
response
Organismal functions signaling
Tissue development Organ morphology LXR/RXR activation
Cell-mediated immune response Embryonic development Coagulation system
Endocrine system development Extrinsic prothrombin
and function Activation pathway
FIG. 2. Venn diagrams showing the overlaps in top biological functions and canonical pathways
associated with the literature, MAP, and LC–MS studies.
Three other canonical pathways were shared between the literature and MAP
studies including ‘‘hepatic fibrosis’’ (Fig. 3B1), ‘‘communication between innate
and adaptive immune cells’’ (Fig. 3B2), and ‘‘role of cytokines in mediating commu-
nication between immune cells’’ (Fig. 3B3). The DR biomarker panel extracted from
the literature was most significantly associated with ‘‘hepatic fibrosis,’’ ‘‘glucocor-
ticoid receptor signaling,’’ and ‘‘atherosclerosis signaling’’ (Table VI).
IV. Discussion
FIG. 3. (Continued)
CONVERGING EVIDENCE OF BLOOD-BASED BIOMARKERS FOR SCHIZOPHRENIA 119
FIG. 3. Canonical pathways most significantly associated with the diagnostic biomarkers identified
from the (A) literature, MAP, and LC–MS studies, and (B1–B3) literature and MAP studies. Canonical
pathways represent graphical representations of the molecular relationships between molecules.
Molecules are represented as nodes, and the biological relationship between two nodes is represented
as an edge (line). All edges are supported by at least one reference from the literature, from a textbook,
or from canonical information stored in the Ingenuity Pathways Knowledge Base. The intensity of the
node color indicates the degree of increased (red) or decreased (green) molecular levels. Nodes are
displayed using various shapes that represent the functional class of the gene product. Edges are
connected by various line connectors that describe the nature of the relationship between the nodes
(see legend; this explanatory extract has been copied from IngenuityÒ Systems; www.ingenuity.com,
with permission). (A) Acute phase response signaling, (B1) hepatic fibrosis, (B2) communication
between innate and adaptive immune cells, (B3) role of cytokines in mediating communication
between innate and adaptive immune cells.
Table VII
COMBINED FINDINGS FROM THE LITERATURE (REFERENCES LISTED IN TABLE) AND IN-HOUSE STUDIES.
Gene # Times Total References Gene # Times Total References Gene name # Times Total References
name found to name found to found to
be be be
altered altered altered
A2M "2 #1 3 Domenici et al. (2010) FABP3 "1 – 1 – MMP2 – #1 1 Domenici et al. (2010)
ACPP "1 #1 2 Domenici et al. (2010) FASLG – #1 1 – MMP7 "2 – 2 –
ACTN2 – #1 1 – FBLN1 – #1 1 – MMP9 "1 – 1 Domenici et al. (2010)
ADIPOQ – #3 3 Bai et al. (2009), Domenici et al. FGA "1 #2 3 Domenici et al. (2010) MPO "2 – 2 –
(2010), Richards et al. (2006)
AGER – #1 1 – FGF2 "1 #1 2 Hashimoto et al. (2003) MUC16 "2 – 2 Domenici et al. (2010)
AGT "1 – 1 – FOLH1B – #1 1 – MYOF – #1 1 –
AHSG – #3 3 Levin et al. (2010) FSHB "1 #1 2 Konarzewska et al. (2009) NAA15 "2 – 2 –
ALB – #2 2 Huang (2002), Yao FTH1 "3 – 3 – NCAM "1 – 1 Tanaka et al. (2007)
et al. (2000)
ANGPT2 "1 – 1 – GH1 – #2 2 Domenici et al. (2010) NGF – #4 4 Jockers-Scherubl et al. (2006),
Lee and Kim (2009), Parikh
et al. (2003)
APCS "3 – 3 Domenici et al. (2010) GHRL – #1 1 Hosojima et al. (2006) NRXN1 "1 – 1 –
APOA1 – #5 5 Domenici et al. (2010), GOT1 "1 – 1 – OXT – #1 1 Keri et al. (2009)
Levin et al. (2010), Yang et al. (2006)
APOA2 – #2 2 Levin et al. (2010) GPX "1 – 1 Atmaca et al. (2005) PCIF1 "1 – 1 –
APOA4 "1 #2 3 Levin et al. (2010), Yang et al. (2006) GREB1 – #1 1 – PDGFB "1 – 1 –
APOC1 – #2 2 Levin et al. (2010) GSN – #1 1 – POLQ "1 – 1 –
APOC3 "1 #2 3 Domenici et al. (2010) GSTA1 "5 #1 6 Domenici et al. (2010) POLR3A – #1 1 –
APOD "2 #2 4 Levin et al. (2010), Mahadik et al. (2002) HFM1 – #1 1 – PPRC1 "1 – 1 –
APOH "5 – 5 Domenici et al. (2010) HP "7 – 7 Yang et al. (2006) PPY "4 – 4 –
APOL1 "1 – 1 – ICAM1 "4 #2 6 Domenici et al. (2010), Kronig PRL "13 #8 21 Berwaerts et al. (2010), Chang et al.
et al. (2005), Schwarz et al. (1998) (2008), Chen et al. (2009), Costa
et al. (2007), Kane et al. (2009),
Kim et al. (2002), Kinon et al. (2006),
Konarzewska et al. (2009),
Kwon et al. (2009), Melkersson
et al. (2001), Montgomery
et al. (2004), Segal et al. (2004, 2007a,
b),
Wang et al. (2007b), Young et al.
(2004),
Zhang et al. (2002a)
AXL – #1 1 – IFNG "1 #1 2 Kim et al. (2004), Na and Kim PROS1 "1 – 1 –
(2007)
B2M "2 – 2 Chittiprol et al. (2009) IGF1 – #2 2 Melkersson et al. (1999), PXDN – #1 1 –
Venkatasubramanian
et al. (2007)
BDNF "7 #18 25 Chen da et al. (2009), Domenici et al. (2010), Gama et al. IGFBP1 – #1 1 Melkersson et al. (2000) RAGE "2 – 2 Steiner et al. (2009)
(2007), Grillo et al. (2007), Guimaraes et al. (2008), Huang
and Hung (2009), Huang and Lee (2006), Ikeda et al.
(2008), Jindal et al. (2010), Lee and Kim (2009), Palomino
et al. (2006), Pirildar et al. (2004), Reis et al. (2008), Rizos
et al. (2008), Tan et al. (2005), Toyooka et al. (2002),
Vinogradov et al. (2009), Xiu et al. (2009), Zhang et al.
(2008b)
BTC "1 – 1 – IGFBP2 "1 – 1 RBP4 – #1 1 –
C3 "4 – 4 Domenici et al. (2010) IL10 "5 #1 6 Domenici et al. (2010), Maes et al. RELN "1 – 1 Fatemi et al. (2001)
(2002)
CALCA "1 – 1 – IL12 "1 – 1 Crespo-Facorro et al. (2008) RETN "2 #1 3 –
CAT "1 – 1 Atmaca et al. (2005) IL12A "1 – 1 Domenici et al. (2010) RGPD1 "1 – 1 –
CCL11 "2 – 2 Domenici et al. (2010), Teixeira et al. (2008) IL12B "1 #2 3 Domenici et al. (2010) S100A12 "2 – 2 –
CCL2 "2 – 2 Domenici et al. (2010), Drexhage et al. (2008) IL13 "1 #2 3 Domenici et al. (2010) S100B "18 #3 21 Gattaz et al. (2000), Lara et al. (2001),
Ling et al. (2007), Pedersen et al.
(2008),
Qi et al. (2009), Rothermundt et al.
(2001b, 2004, 2007), Sarandol et al.
(2007),
(Continued)
Table VII (Continued)
Gene # Times Total References Gene # Times Total References Gene name # Times Total References
name found to name found to found to
be be be
altered altered altered
(Continued)
Table VII (Continued)
Gene # Times Total References Gene # Times Total References Gene name # Times Total References
name found to name found to found to
be be be
altered altered altered
CST3 "1 – 1 – LEP "8 – 8 Atmaca et al. (2003), Baptista TNF-a "6 #2 8 Garcia-Miss Mdel et al. (2010),
et al. (2007), Domenici et al. Monteleone et al. (1997), Na and
(2010), Hosojima et al. (2006), Kim (2007), O’Brien et al. (2008),
Jow et al. (2006), Melkersson and Song et al. (2009), Theodoropoulou
Hulting (2001); Melkersson et al. et al. (2001)
(2000), Wang et al. (2007a)
CTGF "3 – 3 – LHB "1 – 1 – TNFRSF10C "2 #1 3 –
CXCL1 "1 – 1 – LRRC16A – #1 1 – TNFRSF14 "4 – 4 Domenici et al. (2010)
CXCL5 "3 – 3 Domenici et al. (2010) LTA – #1 1 Domenici et al. (2010) TNFRSF1A "2 – 2 Coelho et al. (2008), Hope et al.
(2009)
DEFA1 "1 – 1 Craddock et al. (2008) LUM – #1 1 – TNFRSF1B "1 – 1 Coelho et al. (2008)
DPP4 "1 – 1 Maes et al. (1996b) MAP2 "1 – 1 – TTR – #1 1 Yang et al. (2006)
EDN1 "1 #1 2 Domenici et al. (2010) MDK – #1 1 Shimizu et al. (2003) UMOD "1 – 1 –
EGF "2 #3 5 Domenici et al. (2010), Futamura et al. (2002), Ikeda MIF "3 – 3 – USP54 – #1 1 –
et al. (2008)
F13B – #3 3 Levin et al. (2010) MMP10 "1 – 1 – VANGL1 – #1 1 –
F3 "1 – 1 Domenici et al. (2010) VCAM1 "1 – 1 Domenici et al. (2010)
F7 "1 #1 2 Domenici et al. (2010) VEGFA "3 #1 4 Domenici et al. (2010)
VWF "5 – 5 Domenici et al. (2010), Hope et al.
(2009)
The numbers indicate the number of times a molecule was found to be altered in abundance.
CONVERGING EVIDENCE OF BLOOD-BASED BIOMARKERS FOR SCHIZOPHRENIA 125
IL6 and TNF-a are proinflammatory cytokines primarily secreted from com-
ponents of the innate immune system (activated monocytes and macrophages;
Siegel et al., 2009). IL6 is the chief factor regulating production of most APPs. The
high frequency in which this protein has been reported to be altered in schizo-
phrenia may therefore explain the changes in levels of APPs often observed in
patients (Gabay and Kushner, 1999). IL6 along with IL1a and TNF-a are cyto-
kines known to induce behavioral, neuroendocrine, and metabolic changes simi-
lar to processes observed in schizophrenia. For example, TNF-a leads to glucagon-
induced hyperglycemia and IL1a activates the pituitary–adrenal system (Gruys
et al., 2005). The link between glucocorticoids and insulin and schizophrenia is
well established. Glucocorticoids enhance the stimulatory effects of cytokines on
the production of APPs (Baumann et al., 1987), whereas insulin decreases their
effects on the production of some APPs (Campos et al., 1994). The roles of IL6 and
TNF-a extend beyond the immune system. These molecules, along with other
cytokines, are involved in a cascade of positive and negative feedback regulation,
interact with hormones and neurotransmitters, and represent the key mediators of
the dynamic interaction between the CNS, immune, and endocrine systems
(Turck et al., 2009).
The question of whether the changes in the levels of APPs observed in
schizophrenia patients contribute as causative factors or simply represent a
manifestation of illness remains unknown. Nevertheless, it is known that when
the relevant receptors are repeatedly triggered by stimuli, APR can become
chronic (Gruys et al., 2005). Whether this is the case in schizophrenia patients
requires further examination, although chronic infection as etiological factor has
been discussed for many years (Siegel et al., 2009).
Taken together, the data also pointed to an activated adaptive immune system
in schizophrenia. This is a specific system involved in higher functions such as
immune memory and the ability to be conditioned. Its cellular component
consists of T- and B-lymphocyte cells and is mainly activated by the type-1
immune system [T-helper-1 (Th1) cells, monocytes/macrophages (M1), and
other cell types] which produce activating immunotransmitters IL2 ("8 and #4
studies), IFN-g ("1 and #1 studies), IL12 ("1 study), IL18 ("2 studies), and TNF-a
("6 and #2 studies; Schwarz et al., 2001; Strous and Shoenfeld, 2006). The
humoral component of the adaptive system is made up of specific antibodies
and is predominantly activated by the type-2 system [T-helper-2 (Th2) cells and
monocytes/macrophages (M2)] which produces IL4 ("2 and #3 studies), IL5 (#3
clinical center), IL6 ("15 and #2 studies), IL10 ("5 and #1 studies), IL13 ("1 and #1
studies), and TGF-b ("1 study) (Mills et al., 2000; Schwarz et al., 2001; Siegel et al.,
128 MAN K. CHAN ET AL.
2009; Strous and Shoenfeld, 2006). The cytokines produced by the Th1 and
Th2 cells antagonize each other while promoting their own type of response
(Schwarz et al., 2001).
Several lines of evidence point to an imbalance between the type-1 and type-
2 immune response in schizophrenia with an overactivation of the type-2 response
and a defective type-1 response (Muller et al., 2004). For example, a handful of
studies have described increased antibody production (type-2 response) in schizo-
phrenia patients suggesting the presence of an autoimmune process, observed in
20–35% patients (Siegel et al., 2009). Increases in Th2 lymphocytes in the blood
have also been reported (Sperner-Unterweger et al., 1999). Although IL6 (repeat-
edly reported to be increased) is primarily secreted from activated monocytes/
macrophages [innate and adaptive responses (M2)], it is also produced upon
activation of type-2 immune response and activates the type-2 response leading
to antibody production. The findings that cortisol and IL10 (Th2 cytokine) levels
were consistently elevated in first-onset drug-naive/free patients (4/5 clinical
centers) and the lack of replicating changes in Th1 cytokines suggest an imbalance
between type-1/type-2 systems. Alterations in cortisol levels in patients have been
repeatedly described and reviewed (Bradley and Dinan, 2010). Cortisol inhibits
the IFN-g response by acting directly on T cells or indirectly through IL12.
Increases in the levels of this active glucocorticosteroid decrease Th1 products
subsequently inducing an imbalance between Th1/Th2 cytokines and a shift to
Th2 response (Pinto et al., 2006). Further, cortisol also acts as a powerful stimulant
of plasma IL10 levels (Dandona et al., 1999). IL10 represents a key regulator of
the immune response as it suppresses Th1-dependent cellular immunity and
promotes Th2-dependent humoral immunity (Moore et al., 1993). Cortisol is
also stimulated by IL6 which is induced by either stress or infectious agents. The
repeated observation of absence of change in the levels of IL6 across the clinical
centers suggests that, rather than infection, stress-related glucocorticoid signaling
processes (demonstrated by increased cortisol levels) might occur in first-onset
patients.
naive schizophrenia patients (Masserini et al., 1990). Treatment was also asso-
ciated with an increase in soluble IL2 receptors (sIL2R) which reflects an increase
of activated IL2 bearing T cells (Muller et al., 1997). Review of the literature
resulted in identification of a panel of 29 immunological biomarkers reported to
be altered in response to antipsychotic medication (Table VIIIA). This highlighted
their potential value as molecular predictors of DR. Seven out of these 29 DR
biomarkers were APPs (IL1RN, IGF1, IL6, TNF, IL1B, IL6R, IL8), suggesting a role
Table VIII
DRUG-RESPONSE (DR) BIOMARKERS.
Gene name # Times found to be altered Gene name # Times found to be altered
(B) 10 DR biomarkers shared between the literature and in-house clinical centers
BDNF – #2 "3 #1
CRP "2 – "2 –
FGF2 – #1 "1 –
FSHB "1 – – #1
ICAM1 "3 – – #1
IL1RN "3 – "2 –
IL6 "1 – "5 #1
IL8 "1 – "1 –
INS "2 – "4 –
PRL "3 – "7 #7
The numbers indicate the number of times a molecule was found to be altered in abundance in the
literature and/or in-house studies.
130 MAN K. CHAN ET AL.
Astrocytes and microglia represent the immunological cells in the CNS. While
microglial cells (derived from peripheral macrophages) primarily secrete type-1
cytokines (e.g., IL12), astrocytes inhibit IL12 and ICAM-1 production and secrete
the type-2 cytokine IL10 (Aloisi et al., 2000; Xiao and Link, 1999). The imbalance
in activation of microglial cells and astrocytes has been proposed to reflect the
type-1/type-2 imbalance in the CNS. Since S100b is a marker of astrocyte
activation, increases in the levels of this circulatory protein have been used as a
sensitive marker of brain damage, astrocyte activation, neural death, or blood–
brain-barrier dysfunction (Lara et al., 2001). Therefore, the repeated finding of
increased levels of S100b ("18 and #3 studies) in schizophrenia patients may
suggest overactivation of astrocytes in the CNS of schizophrenia patients.
comprising groups of highly specific and sensitive disease biomarkers are likely to
be required for delineation of complex neuropsychiatric conditions (see Chapter
‘‘Algorithm development for diagnostic biomarker assays’’ by Izmailov et al.).
Acknowledgments
This research was supported by the Stanley Medical Research Institute (SMRI)
and the European Union FP7 SchizDX research program (grant reference 223427).
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Psychopharmacology (Berl.) 204, 177–184.
ABNORMALITIES IN METABOLISM AND HYPOTHALAMIC–
PITUITARY–ADRENAL AXIS FUNCTION IN SCHIZOPHRENIA
Abstract
I. Introduction
II. Peripheral Metabolic Effects
III. Altered Hormone Secretion in Schizophrenia
IV. Altered Hormone Production in Schizophrenia Pituitaries
V. Evidence for Altered Insulin Signaling in Schizophrenia Brain
VI. Environmental Causes of Psychiatric Illness
VII. Specificity of Molecular Signature for Schizophrenia
VIII. Therapeutic Implications
IX. Special Considerations
X. Conclusions
Acknowledgments
References
Abstract
For decades, evidence has been emerging that the pathogenesis of schizophre-
nia can involve perturbations in metabolic and hypothalamic–pituitary–adrenal
(HPA) axis pathways. Variations in manifestation of these effects could be related
to the differences in clinical symptoms between affected individuals as well as to
differences in treatment response, including the finding that a high proportion of
subjects fail to respond to current antipsychotic medications. Here, we review the
evidence for abnormalities in metabolism and HPA axis regulation in schizophre-
nia. Such studies may prove critical for increasing our understanding of the
multidimensional nature of psychiatric illnesses and for improving the timeliness
and accuracy of diagnosis. Stratification of subjects according to molecular
phenotype reflecting the disease state or trait could help to improve existing
treatments through application of novel personalized medicine strategies and by
the development of much-needed novel antipsychotic agents.
I. Introduction
Insulin, proinsulin, and des 31,32-proinsulin levels were measured using two-site
time-resolved fluorescence assays employing different combinations of monoclonal
antibodies that discriminate between the specific forms of the molecule (Sobey et al.,
1989). C-peptide and chromogranin A were measured using commercially available
immunoassays. All of these molecules were present at significantly elevated levels in
serum and plasma from schizophrenia patients (Fig. 1). The finding that these
changes in insulin-related molecules occurred against a background of relatively
normal glucose levels was consistent with the possibility that at least some of these
patients were insulin resistant at the onset of the disease. This could have important
implications since elevated insulin can have deleterious effects on brain function
(Taguchi et al., 2007). In contrast, no significant differences were found in the same
insulin-related molecules in serum from bipolar disorder patients (Guest et al.,
2010a,b), providing some preliminary evidence that these molecules are not changed
in all neuropsychiatric disorders. However, it will be important to carry out similar
studies using blood samples from subjects with other psychiatric conditions such as
major depressive disorder, anxiety disorders, and autism spectrum conditions to
answer this question with greater certainty.
Brain
Hypothalamus
(−) Growth hormone
(−) Arginine vasopressin
Pituitary (−) Secretagogin
Altered HPA function
(+) Prolactin
Impaired insulin signaling
(+) Chromogranin A
(+) ProACTH
Blood vessels
Circulation
(+) Insulin
(+) Proinsulin
(+) Des 31,32 proinsulin
Pancreatic (+) C-peptide
islet cells (+) Pancreatic polypeptide
(+) Chromogranin A
(+) Cortisol
(+) Chromogranin A
Adrenals
(+) Progesterone
Gonads
FIG. 1. Schematic diagram showing potential effects of insulin resistance on secretion of other
hormones and bioactive molecules over the diffuse neuroendocrine system.
150 PAUL C. GUEST ET AL.
Taken together, these findings suggest that some, but not all, schizophrenia
patients show signs of metabolic perturbations such as insulin resistance and altered
glucose handling during the earliest phases of the disease. This leads to the
possibility that increased output from pancreatic islet b cells (the insulin-producing
cells) is required to maintain normal glycemia in these individuals. Through
analyzes of several cohorts, we found that 50% of the first-onset antipsychotic-
naive patients had elevated insulin, proinsulin, and des 31,32 proinsulin levels.
K-means clustering analysis showed that the frequency of subjects with high levels
of insulin-related molecules was 50% for schizophrenia patients (data not shown),
consistent with estimates based on one epidemiological study of insulin resistance in
schizophrenia patients (Timonen et al., 2009). In contrast, the proportion of control
subjects with high insulin levels was less than 20% (data not shown).
(Iwazaki et al., 2004) and CSF (Landen et al., 1999) of patients with schizophrenia.
Given the wide spectrum of biological activities associated with chromogranin
A-derived peptides, it is possible that some of these could have important physio-
logical consequences in the onset or development of schizophrenia.
All of the other hormones which we found to be altered in schizophrenia are
known to have key roles in metabolism, development, or growth. Previous studies
have shown that pancreatic polypeptide is involved in regulation of energy
balance (Asakawa et al., 2003), prolactin has direct effects on insulin production
and insulin resistance (Ben-Jonathan et al., 2006), and high levels of cortisol
(Nosadini et al., 1983) and progesterone (Kalkhoff, 1982) have been linked to
insulin resistance. Only growth hormone showed decreased levels in schizophre-
nia patients. This supports other studies which have shown that subjects with
growth hormone deficiency are insulin resistant (Murray and Shalet, 2005). The
results of previous studies suggest that the reduced growth hormone levels may be
due to schizophrenia-associated increases in dopamine or serotonin activities,
which regulate the production of this hormone (Kahn et al., 1992).
In separate studies, we also found alterations in expression of the neuropeptide
VGF in CSF from first-onset antipsychotic-naive subjects and in postmortem brains
from schizophrenia patients (Huang et al., 2006). VGF is expressed in neurons in
the central and peripheral nervous systems and in various cells of the diffuse
neuroendocrine system (Levi et al., 1985). It appears to be involved in regulation of
energy balance and synaptic plasticity (Alder et al., 2003; Hahm et al., 1999).
As with chromogranin A and insulin, VGF is synthesized initially as a larger
precursor protein which undergoes processing to a number of bioactive peptides.
Researchers have shown that administration of proVGF or synthetic peptides
corresponding to the C-terminal region of proVGF may have therapeutic poten-
tial. Expression of VGF in primary spinal cord neuronal cultures has been shown
to attenuate excitotoxic injury (Zhao et al., 2008). In addition, central nervous
system administration of a putative 22 amino acid VGF-derived peptide appears
to modulate neuropathic pain (Rizzi et al., 2008), increase energy expenditure,
and decrease diet-induced obesity (Bartolomucci et al., 2006). Interestingly, a
recent study showed that exercise stimulates a neurotrophic signaling cascade
which included increased expression of VGF (Hunsberger et al., 2007). The
authors also showed that infusion of a putative 20 amino acid VGF-derived
peptide produced an antidepressant response in mice (Hunsberger et al., 2007).
These findings suggest that drug discovery efforts centered on VGF-derived
peptides may have therapeutic potential in conditions such as chronic pain,
insulin resistance, or psychiatric disorders.
Another important implication of these findings is that other proteins released
from neuroendocrine secretory granules may be altered in the circulation of
schizophrenia patients. Approximately 100 additional proteins have been identi-
fied in insulin secretory granules (Guest et al., 1991) including the prohormone-
ABNORMALITIES IN METABOLISM 153
Peripheral blood biomarkers are useful in clinical practice because of the ease of
accessibility and the associated minimally invasive sampling procedures and low
costs. However, initial discovery can be made using the tissue of origin for many of
these biomarkers. The pituitary releases several hormones and potentially hundreds
of other bioactive peptides into the peripheral circulation, rendering these ideal for
translation to blood-based assays. In addition, HPA axis function is known to be
perturbed in schizophrenia subjects (Ryan et al., 2004). We carried out an analysis of
postmortem pituitary tissue from schizophrenia patients to identify a schizophrenia-
associated molecular fingerprint using a combination of liquid chromatography
tandem mass spectrometry (LC–MS), two-dimensional difference in-gel electropho-
resis (2D-DIGE), and multiplex immunoassay (Guest et al., 2011). This multiplatform
approach was employed to circumvent any limitations in proteomic coverage of these
methods when used alone. This led to identification of differentially expressed
proteins and small molecules including HPA axis-associated molecules such as
cortisol, proACTH, the AVP precursor, agouti-related protein, growth hormone,
prolactin, and secretagogin, and molecules associated with lipid transport and
metabolism, namely the apolipoproteins A1, A2, C3, and H.
ProACTH is produced by cleavage of proopiomelanocortin (POMC) at
amino acid 179 by the prohormone convertase PC1 (Brunelin et al., 2008).
154 PAUL C. GUEST ET AL.
We found that the level of proACTH was significantly elevated in pituitaries from
schizophrenia patients relative to controls. This is consistent with the finding of
clinical studies which showed increased circulating levels of ACTH in schizophre-
nia patients (Ackland et al., 1983; Walsh et al., 2005). In addition, the finding of
increased levels of proACTH and the concomitant elevation in cortisol supports
the hypothesis that HPA axis hyperactivity may be involved in the pathophysiolo-
gy of the disease (Ryan et al., 2004).
AVP is stored as a larger prohormone called vasopressin–neurophysin
2–copeptin (NP2) in the posterior pituitary until the precursor and proteolyzed
forms of this protein are released into the circulation by regulated exocytosis. As a
consequence, reduced production of NP2 may lead to altered levels of circulating
AVP. The primary role of AVP is in homeostasis and regulation of water, salt, and
glucose in the blood. However, there have been studies which have also linked
abnormal AVP levels to changes in mood and behavior (Heinrichs et al., 2009),
and to psychotic disorders (Goldman et al., 1996; Mai et al., 1993). In addition,
AVP may affect HPA axis sensitivity since there appears to be a positive correla-
tion between the circulating levels of AVP, ACTH, and cortisol in anti-naive
schizophrenia patients (Ryan et al., 2004).
The GLUT1 transporter is a transmembrane protein and therefore not likely
to be released from the pituitary into the peripheral circulation, apart from causes
due to cell damage. However, this molecule has been linked to abnormal glucose
activity in schizophrenia brain areas such as the hippocampus, basal ganglia, and
thalamus, as shown by positron emission tomography analysis (Buchsbaum et al.,
1986; Clark et al., 2001). It is thought that acute prodromal schizophrenia symp-
toms can result from inadequate glucose uptake into brain cells due to deficiencies
in GLUT1 and GLUT3 activities (McDermott and de Silva, 2005). Our finding of
decreased GLUT1 levels in the pituitary of schizophrenia suggests that GLUT1 is
a crucial component of HPA axis function and supports the case that glucose
handling may be involved in the pathogenesis or development of schizophrenia.
In line with the above results, we also found reduced expression of several
members of the apolipoprotein family in postmortem pituitaries of schizophrenia
patients. These findings are consistent with those of other studies on the serum
levels of these proteins (Huang et al., 2008; Yang et al., 2006). Apolipoprotein A1
and A2 facilitate transport of cholesterol and triglycerides in the blood
(Kwiterovich, 2000), apolipoprotein C3 is thought to inhibit hepatic uptake of
triglyceride-rich particles (von Eckardstein et al., 1991), and apolipoprotein H
appears to prevent activation of the intrinsic blood coagulation cascade by
binding to phospholipids on the surface of damaged cells (Schousboe, 1985).
Impairment of low-density lipoprotein oxidation and lipid transport can lead to
atherosclerosis and increased risk of cardiovascular events such as myocardial
infarction and stroke, which are all components of the metabolic syndrome
(Alberti et al., 2006). Changes in the levels of apolipoprotein A1 and C3 are
ABNORMALITIES IN METABOLISM 155
already used as biomarkers for metabolic syndrome, and for prediction of cardio-
vascular risk (McQueen et al., 2008; Onat et al., 2003).
We also found decreased levels of secretagogin in schizophrenia postmortem
pituitaries along with increased levels of the same protein in serum in first-onset
antipsychotic-naive schizophrenia patients. This could be indicative of a relative
depletion of this protein in the pituitary due to increased rates of secretion. The
expression of secretagogin is restricted to distinct neuroendocrine cells with high
levels found in the anterior pituitary, pancreatic islet cells, cerebellum, thalamus,
hypothalamus, and neocortical neuronal subgroups (Gartner et al., 2001). Studies
of secretagogin in pancreatic b-cells suggest that it influences calcium-influx,
hormone secretion, and cellular proliferation (Skovhus et al., 2006). In addition,
secretagogin was found to be decreased in diabetes-prone rat islets exposed to
cytokines (Skovhus et al., 2006). In humans, it has been used as a biomarker for
neuroendocrine tumors (Birkenkamp-Demtroder et al., 2005) and ischemic neu-
ronal damage (Gartner et al., 2001; Montaner et al., 2008). As with secretagogin,
we also found a similar decrease in prolactin levels in this study in schizophrenia
postmortem pituitaries we have identified increased serum levels of this hormone in
antipsychotic-naive schizophrenia patients in separate study (Guest et al., 2011). In
the case of both proteins, the fact that these effects were observed in serum from
first-onset antipsychotic-naive schizophrenia subjects is suggestive of a disease-
related change rather than an effect of chronic drug treatment associated with the
postmortem samples. Moreover, detection of these biomarker candidates in an easily
accessible biological fluid such as blood serum adds to the value of these findings
and may lead to translation of these molecules as peripheral biomarkers for
schizophrenia.
The finding that some, but not all, schizophrenia subjects also have hyper-
insulinemia, insulin resistance, or signs of HPA axis dysfunction is consistent with
the hypothesis that schizophrenia is a heterogeneous disorder comprised of
distinct subtypes (Seaton et al., 2001). In addition, it is likely that environmental
factors come to play in precipitation of the disease. Metabolic perturbations which
occur during gestation, such as malnutrition, diabetes, obesity, or neuroendocrine
stress, can lead to metabolic dysfunction in the offspring, which may be due to
abnormalities in the development of hypothalamic connectivity (Levin, 2006).
Epidemiological studies have shown that prenatal deprivation of nutrients
increases the risk of schizophrenia in the offspring. The most convincing study
for which full records are available concerned the Dutch Hunger Winter of
1944–1945. In this case, a widespread famine occurred due to blockade of
occupied Holland in October 1944. This resulted in food shortages, particularly
protein supply, in 40,000 individuals (Brown and Susser, 2008; Hoek et al., 1998).
A 2.7-fold increase in risk of schizophrenia spectrum disorders occurred in
subjects born in December 1945. This indicated that these individuals were
conceived at the peak of the famine when the protein supply was at its lowest
(Hoek et al., 1998). Similar findings resulted from studies of the Chinese Famine of
1959–1961 (St Clair et al., 2005). In this case, detailed dietary information was not
available, although birth cohorts conceived at the peak of the famine showed a
2.30-fold increased risk of schizophrenia (St Clair et al., 2005).
ABNORMALITIES IN METABOLISM 157
One important question is the specificity of metabolic and HPA axis effects to
schizophrenia, since hyperinsulinemia and metabolic and endocrine abnormal-
ities have been implicated in other neurological and neurodegenerative disorders
such as major depressive disorder (Licinio and Wong, 2003; Rasgon and Kenna,
158 PAUL C. GUEST ET AL.
2005), bipolar disorder (Fagiolini et al., 2005), and Alzheimer disease (Mosconi
et al., 2008). For example, the insulin effector protein GSK3b is thought to be a
major target of lithium, the mainstay of treatment for bipolar disorder (O’Brien
and Klein, 2009). Brain-derived neurotrophic factor (BDNF) levels appear to play
a role in the pathophysiology of several psychiatric disorders including schizo-
phrenia (Green et al., 2010), major depressive disorder (Shimizu et al., 2003), and
bipolar disorder (Machado-Vieira et al., 2007), and treatment with drugs such as
antidepressants increases the levels of BDNF in major depressive disorder patients
(Shimizu et al., 2003). Changes in pituitary volume appear to be common in
nonmedicated psychiatric disorder subjects including those with schizophrenia
(Pariante et al., 2004) and major depressive disorder (MacMaster et al., 2006). Also,
increased levels of cortisol have been noted in most psychiatric conditions and, at
least in the case of the major depressive disorder, this increase may precede the
onset of illness by several years (Goodyer et al., 2000).
Recently, we reported a novel analytical approach for identifying biomarkers
of schizophrenia using expression of serum analytes from first-onset, antipsychot-
ic-naive patients (Cheng et al., 2010). This method identifies molecules which
exhibit coregulated behavior by analysis of the expression data in reproducing
kernel spaces. This approach led to identification of a small cluster of analytes
comprising only insulin, growth hormone, leptin, and cortisol that was capable of
distinguishing first-onset schizophrenia subjects from controls with 80% precision.
Interestingly, this same panel of metabolic markers was less strongly related to
major depressive disorder and bipolar disorder. As there is a wide continuum of
symptoms between many of the psychotic disorders, as well as between various
manifestations of schizophrenia and schizophrenia-like conditions, further inves-
tigation of this point would be of interest for increasing our knowledge of these
disorders and for improving diagnosis.
The finding that hyperinsulinemia may play a role in the onset of schizophre-
nia suggests that drugs which improve insulin signaling represent a potential novel
treatment strategy. Existing antipsychotic drugs are capable of inducing metabolic
side effects including insulin resistance, weight gain, and type 2 diabetes (Koller
and Doraiswamy, 2002; Meyer et al., 2008). Interestingly, the degree of weight
gain induced by clozapine and olanzapine has been associated with improved
psychopathology ratings, suggesting that these effects may be related at the
biological level and that changes in these pathways may be essential for therapeu-
tic efficacy (Czobor et al., 2002; Meltzer et al., 2003). In one study, alterations in
ABNORMALITIES IN METABOLISM 159
body weight, blood glucose, and leptin levels were associated with improvement in
positive and negative symptoms (Small et al., 2004) and another study found that
the metabolic abnormalities in CSF were normalized by antipsychotic treatment
(Holmes et al., 2006).
Therapeutic strategies which target the underlying metabolic dysfunction may
provide an effective alternative to treating the traditional neurotransmitter-related
endpoint of the disorder. The insulin-sensitizing agents Metformin and Rosigli-
tazone have been used to correct the antipsychotic-induced insulin resistance
typically associated with antipsychotic treatment without compromising the psy-
chotropic benefits (Bahtiyar et al., 2007). Taken together with our finding of
increased circulating levels of insulin-related peptides in patients free of antipsy-
chotic medication suggests that the use of insulin-sensitizing agents alone might
have some therapeutic benefit. Moreover, this suggests that insulin-related
molecules would have utility as biomarkers not only for patient selection but
also for monitoring responses or side effects of therapeutic treatment strategies.
In addition, these results suggest that initiation and maintenance of treatment
should include routine surveillance for clinical and/or biochemical evidence
suggestive of the metabolic or syndrome or HPA axis dysfunction.
Similar strategies are already proving fruitful for treatment of memory deficits
in Alzheimer’s disease. Clinical trials are focusing on the use of PPAR-gamma
agonist such as Rosiglitazone and Pioglitazone as an alternative therapy to
enhance cognition (Sato et al., 2009). One group conducted a 6-month, rando-
mized, open-controlled trial in patients with mild Alzheimer disease accompanied
with type 2 diabetes (Landreth et al., 2008). They assigned patients to two groups.
One group was treated with 15–30 mg Pioglitazone daily (n ¼ 21) and the other
was used as a control (n ¼ 21). The Pioglitazone group showed improved cogni-
tion and increased regional cerebral blood flow in the parietal lobe, while the
control group showed no such improvements. PPAR-gamma is required for
adipocyte differentiation and fat deposition, and modulates plasma leptin levels,
insulin sensitivity, and glucose homeostasis, although its role in the central nervous
system is not fully understood. It was interesting that the effects of Pioglitazone
were accompanied by changes in cerebral blood flow as such an effect could
potentially lead to increased glucose availability in the brain.
The IDE is a target of PPAR-gamma signaling and has also been proposed
as an alternative therapeutic target for Alzheimer disease (Landreth et al., 2008).
In addition, the development of cognitive enhancers for Alzheimer disease,
based on the inhibition of insulin-regulated aminopeptidase, has been proposed
(Albiston et al., 2007; Chai et al., 2008). This enzyme has both central
and peripheral effects and again provides an example of how peripheral
biomarker discovery can provide novel therapeutic strategies for the treatment
of psychiatric disorders.
160 PAUL C. GUEST ET AL.
X. Conclusions
development. In turn, this could lead to improved diagnosis and patient stratifi-
cation for personalized medicine strategies. Given the potential of this line of
research to improve diagnosis and create alternative treatment strategies, more
research is warranted. Future studies should address the metabolic and HPA axis
dysfunction in subtypes of schizophrenia as well is in other neuropsychiatric
conditions. In addition, longitudinal studies should be performed to investigate
the status of metabolic and HPA axis factors over the course of the disorder and
with antipsychotic treatment. This process will be facilitated by improved molec-
ular technologies and rapid translation of biomarkers into clinical protocols.
Acknowledgments
This research was supported by the Stanley Medical Research Institute (SMRI)
and the European Union FP7 SchizDX research program (grant reference 223427).
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IMMUNE AND NEUROIMMUNE ALTERATIONS IN MOOD
DISORDERS AND SCHIZOPHRENIA
Abstract
I. Introduction
II. Inflammatory Cytokines in Psychiatric Disorders—State of the Art 2010
III. Circulating Immune Cells in Psychiatric Disorders—State of the Art 2010
A. Numbers of Circulating Monocytes
B. Numbers of T Cells and T Cell Subsets
C. Activation of Circulating Immune Cells
IV. Our Recent Studies on the Inflammatory State in Bipolar Disorder and Schizophrenia
A. Proinflammatory Gene Expression
B. Pro- and Anti-Inflammatory CD4þ T Cell Subpopulations
C. Pro and Anti-Inflammatory Monocyte/Macrophage and T Cell Cytokines/
Chemokines
D. Summary of Findings
V. Communication of Immune System and Brain in Psychiatric Illness: The Role of Microglia
A. Altered Inflammatory Set Point of the Brain
B. Alterations in the Tryptophan Breakdown Pathway
VI. The Origin of the Activated Immune System in Psychiatric Patients: Genes or
Environment?
A. The Effect of Genes on the Immune Activation
B. The Monocyte Inflammatory Gene Fingerprint: Environmental Effect?
C. Environmental Factors
VII. Conclusions
Acknowledgments
References
Abstract
A large number of publications over the past 20 years have indicated that
immune system function is altered in schizophrenia and mood disorder patients.
This chapter reviews the evidence, which suggests that a proinflammatory state of
the cytokine network induces psychopathologic symptoms and may be involved in
the pathogenesis and pathophysiology of these major mental illnesses. The
authors also present recent data, which relates immune activation to present
theories on the influence of activated immune cells in altering brain function.
They also focus on the role of the environment in immune activation and on the
role of the microbiome and gut flora. Increased understanding of such factors
could help in the development of novel treatment strategies and improved clinical
management of mental disorders.
I. Introduction
Mast cell
Basophils
Immunoglobulins
Neutrophils
Eosinophils
Granulocytes
Hemopoietic stem cell
B cell
Plasma cell Monocyte
CD4+ CD25+
regulatory T cell
Lymphoid stem cell
Macrophage
IL-1
CD8+ cytotoxic T cell IL-6
TNF-a
TH1 cell
IL-12
IFN-g
Natural killer cell
FIG. 1. The innate and adaptive immune system. Schematic diagram showing the important
components of the innate and adaptive immune systems. Further information is provided in the text.
IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; TGF, tumor growth factor; Th, T helper
cell.
In this chapter, we first give a synopsis of the literature on the reported levels of
cytokines/chemokines in the serum of bipolar disorder and schizophrenia patients
and relate findings to what is known on the actual numbers and activation state of
immune cells in bipolar disorder and schizophrenia. Thereafter, we give some
recent data of ours, summarizing the state of the art on the involvement of immune
cells in major mental disorders and relate immune activation to present theories on
the influence of activated immune cells in altering brain function. Finally, we
discuss the role of the environment on immune activation in psychiatric disorders
and focus in on the role of the microbiome and the dietary/gut flora.
(Continued)
Table I (Continued )
References are indicated between brackets. #: downregulation, ": upregulation, ¼: normal level. The summary columns reflect our conclusions based on this
literature. We concluded that there is an upregulation of IL-1, Il-6, and TNF on the basis of sufficient studies of which approximately half show upregulation and
none showed a downregulation. We concluded that IL-2 is putatively upregulated in schizophrenia because there are sufficient studies of which the cumulative
index shows some upregulation. n.c. means not conclusive due to low number of studies and/or conflicting outcomes.
IMMUNE AND NEUROIMMUNE ALTERATIONS IN MOOD DISORDERS 175
bipolar disorder. Of the cytokine networks, particularly the IL-1, IL-6, and TNF
cytokine networks seem to be activated. There is also some evidence in the literature
that the IL-2 system is activated in schizophrenia. In bipolar disorder, the IL-2
system might be activated during a manic episode. But as indicated above, the data
show in particular that measurements of cytokines in plasma or serum are not
consistent and precise enough to reliably detect an activation of the inflammatory
systems in individual patients or in small groups of patients.
This section examines the question of whether there are also indications of an
activated inflammatory response system on the level of circulating immune cells in
schizophrenia and bipolar disorder.
There are early reports showing that the number of circulating monocytes is
aberrant in schizophrenia. Rothermundt et al. (1998) reported a slight increase in
the mean absolute and relative monocyte counts. (Zorrilla et al. (1996) supported
these observations showing, respectively, a monocytosis and a higher number of
CD14þ cells (CD14 is a marker of monocytes) in nonmedicated schizophrenia
patients. Another study, however, refuted this finding of monocytosis, although this
was using a small sample of patients (Torres et al., 2009a). In the cerebrospinal fluid of
patients with schizophrenia, monocytes and macrophages show an accumulation
during acute psychotic episodes (Nikkila et al., 1999). For bipolar disorder, there is only
one report from our group on numbers of circulating monocytes in bipolar disorder
(Padmos et al., 2008a) and this was focussed on numbers of mature (CD14þCD16þ)
and immature (CD14þCD16neg) circulating monocytes. We did not find abnormal
numbers of these subpopulations in the circulation of bipolar patients. Recently,
we confirmed the higher presence of circulating CD14þ monocytes in schizophrenia
(Drexhage et al., 2011a), but were again unable to detect a higher number
of circulating CD14þ monocytes in bipolar disorder (Drexhage et al., 2011b).
References are indicated between brackets. #, downregulation; ", upregulation; ¼, normal level; (#), downregulated but low number of patients. The summary
columns reflect our conclusions based on this literature. We concluded that there are raised numbers of monocytes in schizophrenia because the cumulative
index of three studies shows upregulation (admitted there are too few studies). We concluded that there are normal-to-putatively raised numbers of CD8þ T cells
in schizophrenia because the cumulative index of five studies shows weak upregulation to normal numbers of CD3 and CD4 T cells and the cumulative index of
eight studies shows normal numbers of cells. n.c. means not conclusive due to low number of studies and/or conflicting outcomes.
178 ROOSMARIJN C. DREXHAGE ET AL.
lymphocyte subsets are normal (Breunis et al., 2003; Cazzullo et al., 1998;
Craddock et al., 2007; Henneberg et al., 1990; Kronfol and House, 1988; Maino
et al., 2007; Muller et al., 1998; Rothermundt et al., 1998; Sperner-Unterweger
et al., 1999; Theodoropoulou et al., 2001; Torres et al., 2009a,b; Zhang et al., 2005,
2009; Zorrilla et al., 1996), although there might be some indication that cytotoxic
CD8þ T cells (particularly naive CD45RA CD8þ T cells) are increased in
schizophrenia (Zorrilla et al., 2001).
IV. Our Recent Studies on the Inflammatory State in Bipolar Disorder and Schizophrenia
Table III
THE PREVALENCE OF SUBCLUSTERS IN BIPOLAR PATIENTS, SCHIZOPHRENIA PATIENTS, AND
HEALTHY CONTROLS.
Positive ¼ mRNA expression > 1standard deviation from the mean level in controls. Signature ¼
positive/negative of the indicated the transcription factors. P values are obtained from Chi-squared test.
a
P < 0.05 versus healthy controls.
b
P < 0.05 versus bipolar disorder.
We extended the previous findings on the IL-2 system and on CD25þ T cells
(see Table I) and confirmed that the IL-2 system is activated in patients with
schizophrenia and bipolar disorder: Also in the new series, we found higher serum
sCD25 (¼ sIL-2R) levels as well as higher percentages of CD25þCD4þT cells in
patients with recent onset schizophrenia and in patients with bipolar disorder,
albeit only for patients younger than 40 years of age in the latter group. It is also of
note that we were only able to carry out FACS analysis for 38 (mean age
41.1 years, 76% female) of the 56 patients with bipolar disorder, all of these
were in a euthymic phase of their disease. At an earlier occasion, we found that the
sCD25 level was highest in cases with an active mania.
We concluded from these recent data that there are clear signs of an activation/
proliferation of the CD4þ T cell compartment particularly in young and active cases
of schizophrenia and young cases of bipolar disorder. However (as we noted in the
previous section), it is not known whether we are dealing here with an activation/
proliferation of the T effector or the T regulatory cell populations. For that, intracel-
lular FACS staining of T cells for IFN-g (Th1), IL-4 (Th2), IL17A (Th17), and
FOXP3 (natural T regulator cells) should be undertaken, which we performed.
We found that the percentage of anti-inflammatory CD4þCD25highFOXP3þ
natural regulatory T cells was higher in the circulation of young and active recent
onset schizophrenia cases and in euthymic bipolar patients of less than 40 years of
age. In addition, we found for schizophrenia that those patients who had the highest
global assessment of functioning (GAF) scores at discharge were the ones with the
highest levels of anti-inflammatory natural T regulator cells at admission. We there-
fore hypothesized that high levels of CD4þCD25highFOXP3þ natural regulatory
182 ROOSMARIJN C. DREXHAGE ET AL.
T cells are probably beneficial and counteract the proinflammatory action of the
monocyte/macrophage system, since it is known that natural T regulator cells are
capable of dampening down the activity of proinflammatory monocytes/macro-
phages. However, it must be noted that we were unable to find a significant correla-
tion between the proinflammatory gene expression in monocytes and the number of
natural T regulator cells. Therefore, this does not suggest a mutual relationship but
rather separate activation mechanisms for monocytes and natural T regulator cells.
In the case of the CD4þ T effector populations, we only found higher
percentages of Th17 cells in our group of young and recent onset schizophrenia
patients. These percentages were normal in the bipolar disorder group (also in
those younger than 40 years).
With regard to the T cell cytokines, we were unable to find any abnormalities
in the serum levels of IFN-g, IL-4, IL-17, IL-10, and TGF-b from patients of both
the study cohorts of recent onset schizophrenia patients and bipolar patients. We
thus conclude that there probably is a balance within an activated T cell system
between pro and anti-inflammatory forces.
In the case of the proinflammatory monocyte/macrophage cytokines/chemo-
kines, we found the serum levels of IL-1b, PTX3, and CCL2 slightly (but signifi-
cantly) raised in bipolar patients and in schizophrenia patients, although this was
only in older patients (mean age 40 years, range 18–65 years) with a chronic form
of the disease. The levels were not raised in the group of young recent onset
schizophrenia patients (mean age 27, range 17–59 years). It is of note that 35% of
the older schizophrenia patients suffered from metabolic syndrome (visceral
obesity, hypercholesterolemia, and diabetes) and that proinflammatory cytokines
were higher in patients with these comorbidities. It must, however, be noted that
proinflammatory cytokines were also raised in the circulation of chronic schizo-
phrenia patients without metabolic syndrome.
We therefore concluded that proinflammatory cytokines are to a limited extent
raised in patients with bipolar disorder and schizophrenia, although this was incon-
sistent. This suggests that the balance within the monocyte/macrophage system
tends to tip toward an active proinflammatory state in these psychiatric diseases.
With regard to the metabolic syndrome, white adipose tissue (WAT) can be seen
as an endocrine organ, and in visceral obesity, WAT contains increased numbers of
tissue macrophages, which are in a chronically inflamed state overproducing various
cytokines including proinflammatory cytokines (in this situation also called adipo-
kines) (Dahlman et al., 2005). Apart from leptin, adiponectin, and PAI-1, CCL2 is
considered an important adipokine. CCL2 promotes migration and accumulation of
IMMUNE AND NEUROIMMUNE ALTERATIONS IN MOOD DISORDERS 183
macrophages into WAT (Trayhurn et al., 2006) and thus supports the further
inflammatory state of WAT. In addition, it induces insulin resistance and therefore
plays an important role as an intermediate in the development of type 2 diabetes in
obese individuals (Cohen et al., 2006; Weigelt et al., 2011). In line with this view, the
levels of CCL2 are higher in patients with visceral obesity and diabetes. Other
proinflammatory cytokines produced by WAT macrophages are TNF-a and IL-6.
To correct for visceral obesity and the metabolic syndrome, the body mass index, the
waist–hip ratio, and levels of HDL need therefore to be taken into account in studies
of proinflammatory cytokines in psychiatric patients.
D. SUMMARY OF FINDINGS
Taken together, the data reviewed here can be interpreted as showing that the
immune system in schizophrenia and bipolar disorder patients is set at a high
activation set point involving both pro- as well as anti-inflammatory forces (see Fig. 2):
1. At the level of the monocyte/macrophage system overlapping but distinct
gene expression patterns (mRNA transcript fingerprints) were found in the
circulating monocytes of patients with schizophrenia and bipolar disorder.
In the serum of bipolar disorder patients, gene transcript cluster 1 and
cluster 2 were upregulated, while in schizophrenia patients, only cluster 1
gene transcripts were upregulated.
A hypothetical interaction model of the fingerprint genes is given in Fig. 3,
showing cluster gene transcripts leading to a proinflammatory action (e.g., IL-1b,
TNF, and CCL2) as well as cluster transcripts which give rise to an anti-inflammatory
action (e.g., ATF3 and NAB2). It is thus not surprising that for the proinflammatory
cytokines monocyte gene expression and circulating protein levels did not reach the
same high levels: For example, in our studies, the approximate sixfold increased
expression of IL-1b at the monocyte mRNA level in bipolar patients was reflected in
only a twofold raised IL-1b protein level in the serum. In our studies on recent onset
schizophrenia patients, the serum levels of proinflammatory cytokines were not
raised at all, while there was a high gene transcript expression in circulating mono-
cytes. Clearly, a regulation at the transcription level is operative in the ‘‘inflammatory,
angry’’ monocytes of bipolar and schizophrenic patients to ensure a close-to-normal
(but still somewhat raised) protein production. The question arises which environ-
mental or endogenous conditions will create a failure of these ‘‘angry’’ monocytes to
keep control over their aberrant gene transcript expressions avoiding a high actual
production of the proinflammatory compounds. Psychological stresses (both acute
and chronic) might be conditions which could lead to this (via adrenaline signaling).
Indeed, stressors have regulating stimulatory effect on IL-1b and IL-6 protein
production (see later).
184 ROOSMARIJN C. DREXHAGE ET AL.
Circulation
sCD25 Gene cluster 2 IL1b sCD25
Gene cluster 1 IL-2 Gene cluster 1 PTX3 IL-2
Monocytosis
Brain Inflammatory Inflammatory
microglia Abnormal microglia Abnormal
development development
and function and function
TH17 ?
ACTH ACTH
Adrenal GC Adrenal GC
gland gland
FIG. 2. Cartoon on immune mechanisms as set in recent-onset schizophrenia and bipolar disorder.
Monocytes in circulation have an activated ‘‘angry’’ inflammatory transcriptome, which differs between
bipolar disorder (clusters 1 and 2) and schizophrenia (cluster 1). Twin studies indicate that cluster 1
overexpression is determined by environmental factors, whereas genetic factors determine for a large
part gene cluster 2 overexpression (see text). It can be envisaged that activated ‘‘angry’’ monocytes, upon
arrival in the (emotional) brain as activated microglia, will display abnormal interactions with neurons
and deregulate synaptic function and neuronal sprouting, and are perhaps even cytotoxic (see text). This
will lead to vulnerability for psychiatric behavior. Also serum cytokines (raised to a certain extent in
patients, reflecting activation of the monocyte/macrophage system) may penetrate the brain and
aggravate the neuronal deregulations of the brain. It can also be envisaged that activated monocytes,
when differentiated to aberrant dendritic cells in the tissues and after having traveled to the draining
lymph nodes, will abnormally stimulate T cells in the secondary lymphoid tissues, such as lymph nodes,
spleen, and lymphatic tissues in the mucosa of the gut and airways. There are signs of an abnormal
differentiation of monocytes to dendritic cells in bipolar disorder patients and these cells have a reduced
T cell stimulatory capacity (see text). With regard to expansion of T cells in secondary lymphoid tissues,
such cells will recirculate and appear in the circulation. In schizophrenia patients, both Th17 cells and T
regulator cells are overrepresented, whereas in bipolar patients, only T regulator cells are more
numerous. As a sign of higher T cell proliferation, sCD25 is higher in the serum of both recent onset
schizophrenia and bipolar disorder patients. As there is a stimulation of both the anti- and proinflam-
matory monocyte and T cell forces, a delicate balance is kept within the system.
FFA RETINOL
STX1A
DHRS3
CDC42
Retinal
Cell
membrane TNF-a
FIG. 3. Hypothetical scheme on interaction of the various fingerprint genes, shown in Table III.
Cluster 1A genes
ATF3 Transcription factor, negative regulator of proinflammatory cytokine expression
DUSP2 Phosphatase involved in the regulation of MAPKinase activity and controlling
inflammatory responses
PDE4B Regulation of inflammatory signal transduction via the c-AMP pathway, defects in the
gene elicit psychosis
IL-6 Inflammatory cytokine
IL-1b Inflammatory cytokine
TNF Inflammatory cytokine
PTX3 Inflammatory compound
PTGS2 Prostaglandin synthase, involved in production of the inflammatory compound PGE2
CCL20 A homeostatic and inflammatory chemokine
CXCL2 Specialized monocyte arrest chemokine promoting adherence of monocytes to
endothelium
CXCL3 Specialized monocyte arrest chemokine promoting adherence of monocytes to
endothelium
EREG Epiregulin, member of the epidermal growth factor (EGF) family, an Il-6 inducing
factor and a downregulating factor for inflammation
TNFAIP3 Essential negative regulator of inflammation, antiapoptotic and inducible by
cytokines through NFkB
BCL2A1 Antiapoptotic factor, inducible by cytokines through NFkB
Cluster 1B genes
EGR3 Transcription factor for early growth response, sympathetic nervous development and
immune regulation
MXD1 MAD/MAD1, transcriptional repressor inhibiting cell growth of monocytic cell lines
(Continued)
186 ROOSMARIJN C. DREXHAGE ET AL.
V. Communication of Immune System and Brain in Psychiatric Illness: The Role of Microglia
There is clear evidence from animal models that an activation of the immune
system influences the brain causing an altered behavior. In our view, the best
model in this respect is the ‘‘maternally induced inflammation model,’’ which
highlights several aspects of the immune to brain communication. Several studies
have shown that intraperitoneal injection of a pregnant rodent at late gestational
IMMUNE AND NEUROIMMUNE ALTERATIONS IN MOOD DISORDERS 187
age with lipopolysaccharide (LPS) gives the pups a long-lasting activation of the
immune system together with behavioral changes (Wang et al., 2010). These
behavioral changes include learning disabilities and reduced social behavioral
performances and are often referred to as schizophrenia-like behavior. LPS
treatment of the pregnant rodents is known to elevate not only levels of various
proinflammatory cytokines in the serum, fetal liver, and amniotic fluid of the
pregnant rodent but also that of proinflammatory TNF-a and anti-inflammatory
IL-10 in the fetal brain. These cytokines are thought to be involved in the
abnormal brain development and behavior of the pup. Blocking antibodies
against IL-6 given to the pregnant rodents prevent the later altered behavior in
the pups (Wang et al., 2010).
Interestingly, there is a human correlate to this model. The relation between
prenatal infections and the development of schizophrenia and mood disorders has
been described for a long time. Mothers suffering from influenza in the first
trimester of pregnancy have a seven times higher chance for the development of
schizophrenia in their offspring, and the effect was three times higher for an
infection in the second trimester (Brown and Derkits, 2010). Another study shows
that mothers seropositive for herpes simplex virus (HSV)-2 in pregnancy have a
two times higher chance of schizophrenia development in their offspring (Brown
and Derkits, 2010). Moreover in a cohort study, IgG antibodies to Toxoplasma
were two times higher in mothers who gave birth to a child with schizophrenia
(Brown and Derkits, 2010). In general, these data are interpreted that the micro-
bial pathogens cross the placenta and cause congenital brain anomalies. This has
been proven for Toxoplasma, HSV, rubella, and cytomegalovirus. However, a role
for immune activation, in general, should not be neglected. It has also been shown
that levels of proinflammatory cytokines were higher in the serum of mothers
during pregnancy, which resulted in a child who later developed a psychiatric
disorder (Brown and Derkits, 2010).
There are two main pathways, which have been described by which a
peripherally activated immune system can influence brain development and
function:
VI. The Origin of the Activated Immune System in Psychiatric Patients: Genes or Environment?
to consistently find specific genes linked to the disorders and to replicate findings
for individual studies. The problem is that psychiatric diseases are probably
heterogeneous conditions from a pathogenesis point of view and are not generally
the result of a mutation in a single or a few genes. Large meta-analyses were needed
to complete the GWAS studies and presently a few genetic markers with a limited
risk have been identified. Amongst these markers are the major histocompatibility
complex (MHC) complex in schizophrenia (Stefansson et al., 2009) and the TNF
gene in major depression (Bosker et al., 2011). A new approach is to find molecular
pathways, which are affected in psychiatric disease. This approach is somewhat
more successful and identified in schizophrenia, for instance, an involvement of the
glutamate metabolism pathway and the tumor necrosis factor receptor 1 (TNFR1)
pathway (Jia et al., 2010). These GWAS data thus strengthen our view for a role for
an activated immune system, which interacts with neurons in psychiatric disease,
as discussed above, yet also suggest that the contribution of genetic polymorphisms
to the activation of the monocyte/macrophage and T cell system is limited.
Recently, Padmos et al. (2009) from our group carried out a case–control study
using monocytes from bipolar twins to determine the contribution of genetic and
environmental influences on the expression of the monocyte proinflammatory
gene signature. It was found that the association of the proinflammatory mono-
cyte gene transcript fingerprint with bipolar disorder was primarily the result of
common shared environmental factors. This was in particular evident for the
overexpression of cluster 1 genes, although some of the cluster 2 genes were also
genetically determined. Epigenetic imprinting is the most likely mechanism via
which environmental factors can create long-lasting activation set points of the
immune system of psychiatric patients and via which even fetal/childhood influ-
ences can impact immune functions later in life. Chronic severe stress, such as in
child abuse, has recently been shown to induce epigenetic changes to the gluco-
corticoid receptor gene in the brain (McGowan et al., 2009). Glucocorticoid
resistance is an important factor in T cell activation of psychiatric patients
(Knijff et al., 2006). Epigenetic modulations of important cluster 1 and 2 finger-
print genes therefore deserve further study.
C. ENVIRONMENTAL FACTORS
Environmental candidates that can act as the shared common factors for
immune system aberrancies and concomitant psychiatric disease are stress, diet,
and infections.
IMMUNE AND NEUROIMMUNE ALTERATIONS IN MOOD DISORDERS 191
1. Stress
The prenatal period is an interesting period to study to understand our
twin data, since the environmental factors experienced by the twins in utero are
hypothetically shared. One of the possible environmental factors that are experi-
enced in utero and can influence both the immune system as well as the brain is
prenatally experienced stress. The literature describes that the effect of prenatal
stress on the immune system mostly leads to a reduction of immune function
(Merlot et al., 2008). However, a few studies report on an exaggeration of inflam-
matory function after prenatal stress. (Hashimoto et al. (2001) showed that prenatal
stress in rats led to an increased fever response to LPS. In addition, Laviola et al.
(2004) demonstrated an increase of spleen and brain frontal cortex levels of IL-1b
in prenatally stressed rats. Possible mechanisms behind prenatal-stress-induced
immune alterations are thought to be (1) a direct influence of maternal hormones
and neurotransmitters on the ontogeny of immune cells, (2) an indirect effect via
deregulation of the hypothalamic pituitary adrenal (HPA)-axis in the prenatally
stressed offspring, and (3) via stress mediator induced change in placental function
(Merlot et al., 2008).
With regard to the effect of prenatal stress on the brain, there is increasing
evidence suggesting that exposure to prenatal stress is a risk factor for psychopa-
thology. Prenatally stressed rats, for instance, show higher emotional reactivity,
higher levels of anxiety, and a depressive-like behavior (Abe et al., 2007). In humans,
a low birth weight is considered an index of prenatal stress, and indeed this has also
been shown to be a risk factor for later development of mood symptoms (Costello
et al., 2007). Also, the amount of stress experienced by the mother during pregnancy
was positively correlated to emotional, cognitive, and behavioral problems of the
offspring (Van den Bergh et al., 2005). It is suggested that stress exposure at critical
time points during fetal development may (1) influence the HPA-axis (Lin et al.,
1998), leading to glucocorticoid resistance and hypercortisolism, (2) alter brain
development, and (3) change neurotransmitter systems (Abe et al., 2007; Austin
et al., 2005; Maccari and Morley-Fletcher, 2007; Van den Bergh et al., 2005). All
these events have been implicated in the pathogenesis of mood disorders (Fig. 4).
Stress experienced later in life is able to induce mood symptoms. In rats, it was
shown that chronic mild stress experienced during adulthood elicited depressive-
like behavior (Willner, 2005). And, in children of bipolar patients, major life events
increased the risk to develop a mood disorder (Hillegers et al., 2004). The impact of
stress on the immune system in adulthood has also extensively been researched. It is
a complex interaction in which the HPA-axis and the sympathetic nervous system
play pivotal roles, especially with regards to their neuroendocrine products cortisol
and catecholamines. These two main mediators of stress effects can regulate a
variety of immune functions such as cytokine and chemokine production, the
trafficking of immune cells and their proliferation, differentiation, and effector
192 ROOSMARIJN C. DREXHAGE ET AL.
A B
SERPINB2
TNFAIP3
BCL2A1
DUSP2
PTPN7
MAPK6
DHRS3
DUSP2
RGC32
PDE4B
CDC42
PTPN7
PTGS2
CXCL2
CXCL3
HSP70
MXD1
EGR3
CCL20
NAB2
FABP5
EREG
ATF3
MXD1
EGR3
MAFF
EMP1
CCR2
THBS
NAB2
CCL2
CCL7
PTX3
STX1
ATF3
IL-1b
CD9
TNF
IL-6
F3
DUSP2
DUSP2
ATF3
ATF3
PDE4B
PDE4B IL-6
IL-6 IL-1b
IL-1b TNF
TNF TNFAIP3
TNFAIP3 Subcluster 1A
BCL2A1
BCL2A1 PTX3
PTX3 PTGS2
PTGS2 CCL20
CCL20 CXCL2
CXCL2 EREG
EREG CXCL3
CXCL3
MXD1
MXD1
EGR3
EGR3
F3
F3
MAFF Subcluster 1B
MAFF THBS
THBS SERPINB2
SERPINB2 RGC32
RGC32
PTPN7 PTPN7
NAB2 NAB2
MAPK6 MAPK6
EMP1 EMP1
STX1
STX1 Subcluster 2
DHRS3
DHRS3
CCL2
CCL2
CCL7
CCL7
CDC42
CDC42
FABP5 FABP5
CD9 CD9
HSP70 HSPA1A
CCR2 CCR2
IL6 TNF
F3 TNFAIP3
BCL2A1 DUSP2
EREG ATF3
CXCL2 PDE4B
CCL20 PTGS2
CXCL3 EREG
IL-1b CXCL2
PTGS2 CCL20
DUSP2 IL-1b
PTX3 IL-6
MAPK6 PTX3
ATF3 CXCL3
CDC42 SERPINB2
PDE4B F3
TNFAIP3 CCL7
TNF RGC32
CCL7 MAFF
SERPINB2 THBS
MAFF MXD1
MDX1 BCL2A1
NAB2 CCL2
STX1A CDC42
CD9 MAPK6
EMP1 EMP1
PTPN7 STX1A
RGC32 PTPN7
CCL2 NAB2
THBS EGR3
EGR3 HSPA1A
HSPAIA CCR2
CCR2 CD9
FIG. 4. Heat map of mRNA transcript correlation. The data represent Spearman’s correlation
coefficients, tested on relative mRNA expression in 56 bipolar disorder and 27 schizophrenic patients.
Significant positive correlations (P < 0.05) are in a red scale (darkest red ¼ correlations > 0.60).
IMMUNE AND NEUROIMMUNE ALTERATIONS IN MOOD DISORDERS 193
functions. The final outcome is, although dependent on the quantity and quality of
stress and on coping strategies, an increased susceptibility to infection and inflam-
matory and autoimmune diseases (Leonard, 2006; Padgett and Glaser, 2003).
Significant negative correlations are in a green scale. White ¼ not significant. (A) Correlations
between all tested mRNA transcripts. (B) Two main clusters can be seen (the cluster on the left top
can be divided into subclusters 1A and 1B). Three sets of transcription factors/MAPK regulators were
extracted (DUSP2/ATF3, MXD1/EGR3, and PTPN7/NAB2) and correlations to the other tran-
scripts are shown. DUSP2/ ATF3 correlate strongest to subcluster 1A transcripts, MXD1/EGR3
correlate strongest to subcluster 1B transcripts and many subcluster 1A transcripts and PTPN7/NAB2
correlate strongest to subcluster 2 transcripts. (C) Hierarchical clustering tree, showing relationship of
mRNA transcripts in schizophrenia and bipolar disorder patients.
194 ROOSMARIJN C. DREXHAGE ET AL.
Studies have demonstrated that being born or raised in a city is a risk factor for
developing bipolar disorder. This is thought to be due to household crowding, and
the consequent high exposure to infectious agents (Torrey and Yolken, 1998). Also
relating bipolar disorder to infections are reports that an excess of winter and
spring births resulting in increased incidence of bipolar disorder in the offspring,
as these children are thought to be more prone to develop perinatal infections
(Torrey et al., 1997). Of the infectious agents, viruses are likely candidates (Yolken
and Torrey, 1995). First, viruses are known for their neurotropism and latency.
Second, viral infections can be accompanied by depressive symptoms and manic
behavior. And third, the mood stabilizers lithium and valproate have been
described to have antiviral effects (Amsterdam et al., 1996; Witvrouw et al.,
1997). The viruses mentioned in the literature as being associated with bipolar
disorder are HSV, CMV, and Borna virus (Hinze-Selch, 2002; Taieb et al., 2001;
Yolken and Torrey, 1995). However, the data are inconsistent with regard to the
presence of virus specific antibodies in serum as well as with regard to detecting
virus RNA in brain or in PBMCs of bipolar patients (Hinze-Selch, 2002; Taieb
et al., 2001; Yolken and Torrey, 1995). With regard to bacterial infections, Borrelia
burgdorferi is known to be able to induce symptoms reminiscent of those seen in
bipolar disorder (Fallon and Nields, 1994). Unfortunately, no systematic research
has been done on the prevalence of this bacterium in bipolar disorder. Toxoplasma
gondii, an intracellular protozoan parasite, is capable of latency and brain infiltra-
tion (Carruthers and Suzuki, 2007) and is therefore an interesting candidate to
study in psychiatric diseases. In schizophrenia, many studies have reported a
correlation to Toxoplasmosis infection (Torrey et al., 2007), especially in cases of
prenatal exposure. However, only one such study is available for bipolar disorder
(to the best of our knowledge) and this reports a negative result with regard to the
presence of T. gondii sequences in postmortem brains (Conejero-Goldberg et al.,
2003). Nevertheless, valproate does inhibit T. gondii development (Jones-Brando
et al., 2003), suggesting that more extensive research is needed to determine
whether or not Toxoplasmosis plays a role of significance in bipolar disorder as
has been suggested in the case of schizophrenia.
VII. Conclusions
bipolar disorder. We presented recent data, which relates the immune activation
to present theories on the influence of activated immune cells in altered brain
function. We also focused on the role of the environment in immune activation
and the role of the microbiome and dietary factors in the cause and potential
prevention of psychiatric conditions. Increased understanding of such factors
could help in the development of novel treatment strategies and improved clinical
management of mental disorders. Possibly, a proinflammatory state may not be a
feature of all patients, but may be a predominant feature of a subset of patients.
Further research efforts should be aimed at elaborating this possibility. Indeed,
such a subset of patients within the larger bipolar or schizophrenia syndromes
may benefit from targeted treatment.
Acknowledgments
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BEHAVIORAL AND MOLECULAR BIOMARKERS IN
TRANSLATIONAL ANIMAL MODELS FOR NEUROPSYCHIATRIC
DISORDERS
Zoltán Sarnyai1, Murtada Alsaif2, Sabine Bahn2,3, Agnes Ernst2, Paul C. Guest2,
Eva Hradetzky2, Wolfgang Kluge2, Viktoria Stelzhammer2 and Hendrik Wesseling2
1
Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom
2
Department of Chemical Engineering and Biotechnology, University of Cambridge,
Cambridge, United Kingdom
3
Department of Neuroscience, Erasmus Medical Centre, Rotterdam, The Netherlands
Abstract
I. Introduction: The Problems with Animal Models
II. Toward Etiological Models in Neuropsychiatry
A. Genetic Susceptibility: The Power of the Mutant Mouse
B. Developmental Insults: Setting the Stage for Life
C. The Stress of Life: The Cost of Impaired Adaptation
D. Disrupting Communication: Pharmacological Modification
III. Reverse Translation
IV. Statistical Methods to Link Biomarkers from Animal Models with the Human Disease
V. Integration of Animal Models Using the Framework of RDoC
Acknowledgments
References
Abstract
In the past 10 years, there have been massive advances in the use of genetically
modified mice to study etiological mechanisms involved in psychiatric disorders.
This progress has been driven by rapid developments in molecular biology techni-
ques and has led to a better understanding of the behaviorally and neurobiologically
relevant functions of genes identified through human genetic studies as risk factors.
Transgenic approaches focusing on common polymorphisms, rare structural muta-
tion, and neurotransmission hypotheses are presented in this section (Table I).
1. Common Polymorphisms
Several mutant mouse models for schizophrenia susceptibility genes have
been created and phenotypically characterized including NRG1, DISC1, and
PRODH (Allen et al., 2008). Numerous genome-wide scan studies have identified
neuregulin 1 (NRG1) as one of the most promising candidate genes for schizophrenia
(Stefansson et al., 2002). NRG1 contributes in diverse cellular processes including
neurodevelopment and regulation of synaptic plasticity through N-methyl-D-asparate
(NMDA) receptors and glutamatergic signaling. Mice lacking any one of the several
isoforms of Nrg1 show a variety of behavioral abnormalities in positive and negative
effect domains and in response to novel environment or social novelty (Table I).
Importantly, some of these behavioral abnormalities can be partially reversed by the
atypical antipsychotic clozapine (Wang et al., 2008).
Initially, the DISC1 (disrupted-in-schizophrenia) gene was discovered through
its segregation with mental illness in a Scottish family, and this finding was
replicated in a variety of populations worldwide (Hwu et al., 2003, St Clair et al.,
1990). The functions of DISC1 remains elusive, but a role in neurite outgrowth,
cell migration, and cell signaling is proposed. Several DISC1 mutants have been
created and a natural mutation was discovered in the commonly employed 129S6
Sv/Ev strain, which exhibits a deficit in working memory (Koike et al., 2006).
Table I
BEHAVIORAL PATTERNS IN ANIMAL MODELS FOR NEUROPSYCHIATRIC DISORDERS.
Disease Fear Stress Anhedonia Reward seeking Reward Attention and Memory Social Aggression Arousal and
learning Gating interaction sleep
Genetic susceptibility
Polymorphisms
NRG1 mutants Nrg1(DEGF)þ/ SCZ $ (1) "/# (2) "/# (2, 3) " (1) $ (2) $ (2) # (2)
Nrg1(DTM)þ/ SCZ $ (4) # (5) $ (6) # (4) $ (5) $ (5) # (7) " (7)
Nrg1(DIG)þ/ SCZ $ (8) # (8)
Nrg1 OE SCZ $ (9) # (9) $ (9)
DISC1 mutants mDisc1 trunc SCZ $ (10) $ (10) # (10)
31L Disk SCZ $ (11) $ (11) $ (11) # (11) $ (11) # (11)
100P Disc1 SCZ $ (11) " (11) # (11) $ (11) $ (11)
hDISC (DN) SCZ $ (12) " (14) " (12) # (13) # (13) # (14) # (12) # (12) " (12)
PRODH mutants PRODH KO SCZ $ (15) # (15) # (15) # (15) $ (15)
Grik2 mutant GluR6 KO BD # (16) # (16) " (16) " (16) " (16) $ (16) $ (16) " (16) " (16)
Rare structural mutations
22q11 mutations (VCFS/ Del2Aam SCZ $ (17) # (17) " (17) # (17) # (17)
DiGeorge sy.)
Del1Rak SCZ $ (18) $ (18) $ (18) # (18)
DelAwb SCZ $ (19) $ (19) # (19) $ (19) " (19)
Df1 SCZ # (20) # (20)
(Continued)
Table I (Continued )
Disease Fear Stress Anhedonia Reward seeking Reward Attention and Memory Social Aggression Arousal and
learning Gating interaction sleep
Neurotrasmitter-signaling hypotheses
Dopamine DAT KO SCZ " (21) $ # (23) " (24) # (24) # (25) # (26) # (27) # (28) $/" (29, $/" (29, # Theta
(22) 30) 30) activity (31)
Glutamate NR1-KD SCZ # (32) " (33) # (34) # (35) # (36, 37) # (38) # (38) # (33) # Theta
activity (38)
GSK3b signaling GSK3b[S9A]- BD $ " (39)
OE (39)
clock mutant BD # (40) # (40) " (40) " (40, " (40) # Sleep (40)
41)
Developmental insults
Maternal immune SCZ " (42) $ # (43) " (44) # (42) # (45) # (45) # (43)
activation (42)
Maternal protein SCZ # (46) " (47) # (48) # (49) # (50)
malnutrition
Interruption of SCZ " (51) " (52) # (53) # (54) # (55)
neurogenesis by MAM
Neonatal ventral SCZ $ (56) " (56) # (57) " (56) " (56) # (57) # (58) # (59) # (59) # (60) " sw EEG
hippocampal lesion activity (61)
Stress and impaired adaptation
Social isolation/isolation MDD/ " (62) # (63) $/" #/$ " (66) " (66) " (65) # (66) $/# (62) " (67) " (66)
rearing SCZ (62) (64, 65)
Social defeat MDD " (68) " (69) " (68) " (70) # (69) " (71) # (70) # (69) $ (69) # (68) " sw EEG
activity (72)
Chronic variable stress MDD "/$ " (75) " (75) " (76) " (76) " (77) # (78, # (78, 79)
(73, 74) 79)
Pharmacological modifications
Glutamate hypothesis Phencyclidine— SCZ # (80) " (81) " (82) " (83) # (84) # (85) # (86) # (87) "/$ (82) # (82)
acute
Phencyclidine— SCZ " (81) $ (82) " (88) # (89) # (90) # (82) # (91) " (91)
chronic
Ketamine SCZ " (81) " (92) # (93) # (94) # (95)
MK801 SCZ " (96) " (97) # (98) # (99) # (91)
Dopamine hypothesis - SCZ " (100) " " (100) # (100)
Amphetamine (101)
BD, bipolar disorder; DISC1, disrupted-in-schizophrenia 1; GSK-3b, glycogen synthase kinase b; MAM, methylazoxymethanol; MDD, major depressive disorder; NREM,
non-rapid eye movement sleep; NRG1, neuregulin 1; PRODH, proline dehydrogenase; SCZ, schizophrenia; sw, slow wave; VCFS, velo-cardio-facial syndrome.
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210 ZOLTÁN SARNYAI ET AL.
Alterations in the organization of newly formed and mature neurons and deficits
in short-term plasticity may contribute to cognitive impairment. Behavioral
studies suggest that DISC1 transgenic mice produce a wide range of subtle
schizophrenia-like phenotypes. Variants created by an N-ethyl-N-nitrosourea
(ENU) chemical mutagenesis program showed either depression-like behavior
with deficits in the forced swim test (Q31L exon) or schizophrenia-like behavior
with deficits in prepulse inhibition and latent inhibition (L100P exon; Clapcote
et al., 2007). The Q31L-associated deficits could be ameliorated by the antide-
pressant bupropion, and the L100P deficits improved after antipsychotic treat-
ment (Clapcote et al., 2007).
Chromosome 6q contains the regulatory region for the human Grik2 gene,
which encodes the glutamate receptor 6 (GluR6). A specific haplotype of this
region was found to be associated with BD (McQueen et al., 2005). GluR6 mRNA
levels were lower in the entorhinal cortex of BD patients (Beneyto et al., 2007).
GluR6 knockout (KO) mice showed increased exploratory behavior and hyperac-
tivity, were more responsive to pharmacological stimulants, were more aggressive
to intruders, were more dominant, and displayed hypersexual behavior. In addi-
tion, GluR6 KO mice were less anxious and took more risks and were also less
immobile in the forced swim test, suggesting increased goal directed behavior.
Passive avoidance learning was normal in GluR6 KO mice (Shaltiel et al., 2008).
Chronic lithium chloride treatment reversed the hyperactivity, aggression, and
exploratory behavior in the GluR6 KO mice. These findings map quite well into
the behavioral syndrome associated with BD. It should be noted that GRIK2
abnormalities have also been linked to autism (Strutz-Seebohm et al., 2006) and
obsessive compulsive disorder (Sampaio et al., 2010).
2. Rare Structural Mutations
A complementary approach to look for psychiatric disorders susceptibility
genes is to study cosegregating chromosomal abnormalities, such as the micro-
deletion at 22q11.2 (22g11DS). This deletion causes the velocardiofacial (VCFS)/
DiGeorge syndrome, which presents with congenital abnormalities affecting sev-
eral tissues and organs. About 25% of the 22q11DS patients develop schizophrenia
and schizoaffective disorder (Bassett et al., 2005). Moreover, 22q11DS is found in
2% of patients with schizophrenia (Karayiorgou et al., 1995) and in 6% of cases of
childhood onset schizophrenia (Usiskin et al., 1999). Importantly, genes within this
region such as catechol O-methyl transferase (COMT), proline dehydrogenase
(PRODH), zinc finger, DHHC-type containing 8 (ZDHHC8) and guanine nucleo-
tide-binding protein (G protein), beta polypeptide 1-like (GNB1L) have been
independently associated with schizophrenia. 22q11DS has been either complete-
ly or partially recreated in several mouse models (Table I). Most of these models
display decreased density of dendritic spines and decreased dendritic complexity of
CA1 pyramidal neurons as well as disturbance in prepulse inhibition, fear
BEHAVIORAL AND MOLECULAR BIOMARKERS 211
conditioning, and working memory. These models offer an interesting tool to study
cognitive deficits as 22q11DS patients exhibit cognitive deficits similar to those
observed in schizophrenia such as effects on attention processing and verbal
working memory (Woodin et al., 2001). The 22q11.2 mouse model further offers
the opportunity to understand the biological basis for the increased psychosis risk
associated with this genetic lesion. In addition, models of the 22q11DS are likely to
capture the interactions among the affected genes (Karayiorgou et al., 2010).
3. Hypothesis-Driven Models
The mechanism of action of drugs that provide symptomatic relief in psychi-
atric disorders has played a major role in the development of hypotheses to
understand disease mechanisms. First generation antipsychotics act predominant-
ly as DA receptor antagonists. Pharmacological stimulation of DA neurotransmis-
sion with indirect agonists, such as the psychostimulant amphetamine, induces
psychosis-like states in susceptible individuals. These initial findings have led to
formulation of the DA hypothesis of schizophrenia, which proposes that hyperac-
tive DA neurotransmission in the mesolimbic system may underlie the psychotic
features of the disease (Meltzer and Stahl, 1976). More recently, similar psychosis-
inducing effects via inhibition of glutamatergic transmission, using NMDA gluta-
mate receptor antagonists ketamine and PCP, have given rise to the glutamate
hypothesis. This implicates hypoactive glutamatergic neurotransmission in devel-
opment of the psychotic and cognitive symptoms of schizophrenia (Coyle, 2006a).
The discovery of the effects of lithium on a key signaling molecule downstream of
DA receptors, glycogen synthase kinase 3 beta (GSK3b), has led to novel hypoth-
eses on the pathogenesis of BD (Ikonomov and Manji, 1999).
Mice lacking the DA transporter (DAT) are unable to reuptake DA released
from synaptic terminals, which leads to elevated DA in the synapse. DAT-KO
mice are hyperactive in novel environments, show excessive stereotypic behavior,
and exhibit sleep dysregulation and dwarfism. Therefore, these mice replicate
some of the features of the amphetamine model of schizophrenia (Table I). The
hyperlocomotion effect can be reversed by the DA receptor antagonists haloperi-
dol and clozapine (Spielewoy et al., 2000). Based on the glutamate receptor
hypofunction hypothesis of schizophrenia, a mutant with 90% reduction in
NMDA receptor 1 expression was created. These NMDA receptor hypomorph
mice show motor activity abnormalities and deficits in social and sexual behavior
(Table I). These abnormalities can be normalized with haloperidol and clozapine,
suggesting an effect on DA and glutamate systems (Mohn et al., 1999).
Lithium (Li) is a frontline drug for BD and it is thought to act through GSK3b
inhibition (Marmol, 2008). Therefore, Prickaerts et al. investigated the GSK3b
[S9A]-overexpressing heterozygous mice as a model of mania. These mice dis-
played a number of BD-like features in the open field test, forced swim test, and
acoustic startle response (Prickaerts et al., 2006). In addition, the wet weight of the
212 ZOLTÁN SARNYAI ET AL.
GSK3b[S9A] mice was 15% lower compared to the wild type in spite of no
difference in central nervous system proliferation rates as measured by [3H]
thymidine DNA incorporation (Prickaerts et al., 2006). Adrenocorticotrophic
hormone (ACTH) and corticosterone plasma levels after stress were normal in
these mice (Prickaerts et al., 2006), similar to the normal cortisol levels found in
BD patients (O’Brien et al., 2006). However, hippocampal brain-derived neuro-
trophic factor (BDNF) levels were significantly higher in GSK3b mice (Prickaerts
et al., 2006), unlike in postmortem brain tissue from human BD subjects where this
protein was found to be lower (Grande et al., 2010).
Disrupted circadian rhythm caused by or leading to less sleep often marks the
beginning of a manic attack (Salvadore et al., 2010). The CLOCK protein is integral
to the regulation of the circadian rhythm and is regulated by GSK3b. Further, clock
polymorphisms have been implicated in recurrence of BD episodes (Benedetti et al.,
2003). To this effect, clock mutant mice were studied and showed mania-like
symptoms. These mice are hyperactive (Roybal et al., 2007) and more sensitive to
stimulants in the intracranial self-stimulation model (Roybal et al., 2007) and to
cocaine (McClung et al., 2005); they show a sucrose preference and decreased
anxiety, and require less sleep (Roybal et al., 2007; Easton et al., 2003). Chronic
Li treatment reverses their hyperactivity and decreased anxiety-like behavior
(Roybal et al., 2007). The ventral tegmental area (VTA) is a brain reward region,
and DA activity was found to be heightened in the VTA of clock mutant mice
(McClung et al., 2005). Hippocampal acetylcholine (ACh) levels are lowered in
mutant clock mice (Sei et al., 2003). Interestingly, red blood cell choline levels were
lower in BD patients (Müller-Oerlinghausen et al., 2002). Further, donepezil, an
acetylcholinesterase inhibitor which increases synaptic Ach, has had positive effects
in a small cohort of treatment-resistant BD patients (Burt et al., 1999). Clock mutant
mice express disrupted hepatic levels of Bmal1, aldolase, arginase, and catalase
(Reddy et al., 2006) as found in BD subjects (Kovanen et al., 2010; Yanik et al., 2004).
Genetic susceptibility
Polymorphisms
NRG1 mutants Nrg1(DEGF)+/ SCZ
Nrg1(DTM)+/ SCZ # NMDA receptors;
hyperphosphorylated NR2b (1, 2)
Nrg1(DIG)+/ SCZ
Nrg1 OE SCZ Hypermyelination of small diameter
axons (3)
DISC1 mutants mDisc1 trunc SCZ Abnormal DG neurons (4)
31L Disk SCZ # Brain volume (5) # PDE4B binding and activity (5)
100P Disc1 SCZ # Brain volume (5) # PDE4B binding (5)
hDISC (DN) SCZ " Brain ventricles, #dendritic # PV in CTX, # DISC1, LIS1, SNAP25
complexity (6) (6)
PRODH mutants PRODH KO SCZ # Glu, Asp, GABA in FC (7)
Grik2 mutant GluR6 KO BD # GluR5 and KA2 in HPC (8)
Rare structural mutations
22q11 mutations Del2Aam SCZ # Dendritic complexity in HPC (9) Altered ox.phos. gene expression in
(VCFS/DiGeorge FC/HPC (9)
sy.)
Disturbed dendritic spines, # Glu Altered miRNA biogenesis (10)
synapses (10)
Del1Rak SCZ Compromised neurogenesis in CTX
(11)
Disrupted basal progenitor
proliferation (11)
DelAwb SCZ
Df1 SCZ
(Continued)
Table II (Continued )
Neurotrasmitter-signaling hypotheses
Dopamine DAT KO SCZ " DA tone, # PRL (12, 13) Anterior pituitary hypoplasia (12, 13) # Postsynaptic DRD1/DRD2 in STR
(12, 13)
# ProENK-A and " DYN mRNA in
STR (12, 13)
Glutamate NR1-KD SCZ $ DA content in STR (14)
GSK3b signaling GSK3b[S9A]-OE BD $ ACTH/CORT (15) # Brain weight, $ neurogenesis in " BDNF mRNA in HPC (15)
HPC (15)
clock mutant BD " DA activity in the VTA, # Disrupted Bmal1, aldolase, arginase
ACh in HPC (16, 17) and catalase in liver (18)
Developmental insults
Maternal immune SCZ " Brain and ventricle volume, # HPC Altered DA, 5-HT, Glu and GABA
activation volume (19, 20) markers in the brain (19, 21)
Abnormal layering, neuron
morphology (19)
and synaptic markers in FC and HPC
(19, 20)
Maternal protein SCZ # Brain and HPC volume (22) Altered DA, 5-HT, Glu and GABA
malnutrition markers in the brain (23, 24)
# Cell number in HPC (22)
Interruption of SCZ # Brain, FC and HPC volume (25) Altered DA, Glu and GABA markers in
neurogenesis by the brain (26, 27)
MAM
Abnormal layering and # PV
interneurons in CTX and HPC
(27, 28)
Neonatal ventral SCZ Altered DA and Glu release Altered GABAergic markers in FC (31)
hippocampal lesion in FC (29, 30)
Stress and impaired adaptation
Social isolation/ MDD/ " CORT and TNF-a in # Chandelier neurons in PFC (34) " CRF-R1 mRNA in DR, # BDNF
isolation rearing SCZ plasma (32, 33) mRNA in HPC (35, 36)
" DA in ACB and STR, " NA IL-1b in HPC and CTX (38)
in HPC and CTX (37)
Social defeat MDD " CORT in serum and IL-1b " CRF mRNA in HPC, # BDNF
in plasma (39, 40) mRNA in HPC, FC and AMY (40, 41)
# DA in FCTX, " 5-HT and " TNF-a in splenic dendritic cells (43)
NA in HPC (42)
Chronic variable MDD # DA and 5-HT in FC, HPC " CRF mRNA in PVN, " TNF-a and
stress and STR (44) IL-1b in HYPO (45, 46)
" CORT, IL-1b and TNF-a $ BDNF mRNA in HPC and AMY
in plasma (45) (47)
Pharmacological modifications
Glutamate hypothesis Phencyclidine— SCZ " GABA enzymes in the Impaired cerebral glucose utilization " 5-HT receptors in CTX (50)
acute brain (48) (49)
Impaired Glu metabolism
(51)
Phencyclidine— SCZ " 5-HT and 5-HIAA in STR # LAADC mRNA expression (52)
chronic (48)
# DA utilization in FC (53)
Ketamine SCZ # DA transmission in FC (54) Impaired cerebral glucose utilization " D2 binding in HPC, # Glu binding in
(55) FC (56)
" Neurogenesis, # PV interneurons
(57, 58)
MK801 SCZ " Glu in FCTX (59) Altered 2-DG uptake (55)
" Monoamines in STR (60)
Dopamine hypothesis— SCZ " Cerebral glucose utilization (61) " D2 dimerization, " CaMKIIb mRNA
amphetamine in STR (62, 63)
" Oxidative stress (64)
BD, bipolar disorder; DISC1, disrupted-in-schizophrenia 1; GSK-3b, glycogen synthase kinase b; MAM, methylazoxymethanol; MDD, major depressive disorder;
NREM, non-rapid eye movement sleep; NRG1, neuregulin 1; PRODH, proline dehydrogenase; SCZ, schizophrenia; sw, slow wave; VCFS, velo-cardio-facial
syndrome.
(1) Bjarnadottir et al. (2007), (2) Stefansson et al. (2002), (3) Michailov et al. (2004), (4) Kvajo et al. (2008), (5) Clapcote et al. (2007), (6) Pletnikov et al. (2008), (7) Gogos et al.
(1999), (8) Shaltiel et al. (2008), (9) Mukai et al. (2008), (10) Stark et al. (2008), (11) Meechan et al. (2009), (12) Giros et al. (1996), (13) Gainetdinov et al. (1998), (14) Mohn et
al. (1999), (15) Prickaerts et al. (2006), (16) McClung et al. (2005), (17) Sei et al. (2003), (18) Reddy et al. (2006), (19) Fatemi et al. (1999), (20) Cotter et al. (1995), (21) Meyer
et al. (2008), (22) Marichich et al. (1979), (23) Palmer et al. (2004), (24) Steiger et al. (2003), (25) Moore et al. (2006), (26) Flagstad et al. (2004), (27) Flagstad et al. (2005), (28)
Goto and Grace (2006), (29) Lipska et al. (1995), (30) Stine et al. (2001), (31) Tseng et al. (2008), (32) Sandstrom and Hart (2005), (33) Wu et al. (1999), (34) Bloomfield et al.
(2008), (35) Belmaker and Agam (2008), (36) Meng et al. (2011), (37) Fone and Porkess (2008), (38) Pugh et al. (1999), (39) Marini et al. (2006), (40) Carobrez et al. (2002),
(41) Pizarro et al. (2004), (42) Watt et al. (2009), (43) Powell et al. (2009), (44) Ahmad et al. (2010), (45) Duncko et al. (2001), (46) Grippo et al. (2006), (47) Allaman et al.
(2008), (48) Hsu et al. (1980), (49) Tamminga et al. (1987), (50) Nabeshima et al. (1986), (51) Nishijima et al. (1996), (52) Buckland et al. (1997), (53) Jentsch et al. (1997), (54)
Moghaddam et al. (1997), (55) Miyamoto et al. (2000), (56) Becker et al. (2003), (57) Keilhoff et al. (2004a), (58) Keilhoff et al. (2004b), (59) Kondziella et al. (2006), (60) Ali
et al. (1994), (61) Orzi et al. (1983), (62) Wang et al. (2010), (63) Greenstein et al. (2007), (64) Frey et al. (2006).
BEHAVIORAL AND MOLECULAR BIOMARKERS 217
of negative and positive effect, cognition, and social functions. Although some
behavioral deficits seem to be to reversed through psychiatric medications, few
antipsychotics have been tested to date (Sanberg et al., 1985; Shi et al., 2003).
The most reproducible neuromorphological findings in schizophrenia include
reduced brain weight and hippocampal volume, and enlargement of the ventricles
(Brown et al., 1986; Bogerts et al., 1990). Maternal malnutrition and interruption
of neurogenesis reproduce those features. However, maternal immune activation
mimics changes in cortical and hippocampal morphology, and in synaptic mar-
kers as seen in some schizophrenia patients (Fatemi et al., 2002). Findings of
disturbance in neurotransmitter systems in schizophrenia are somewhat less
robust (Vollenweider et al., 1998). Nevertheless, neurodevelopmental models for
schizophrenia feature DA-, glutamate-, GABA-, and serotonin-related molecular
and functional deficits (Table II).
Stress is one of the most significant risk factors for development and progres-
sion of a number of psychiatric disorders (Nestler et al., 2002). Several animal
models involving early life stress have been used in MDD research including
prenatal stress, postnatal handling, maternal separation, and social isolation at
weaning (Francis et al., 1996; Ladd et al., 2000; Meaney, 2001). Stress can also be
induced later in life by competition within a social environment. Indeed,
humiliating defeats and/or entrapment are associated with a greater risk of
developing depressive symptoms in women (Brown et al., 1995). A relevant animal
model of depression based on naturalistic stressors is social defeat. The chronic
variable stress (CVS) model entails various stressors applied over long time
periods, mimicking the likely situation in humans (McArthur and Borsini,
2006). In the CVS model, stressors include foot shock, cold water immersion,
48-h food and water deprivation, and milder conditions such as heat stress, cage
tilt, reversal of day/night cycle, and change of cage mates (Willner, 1997).
Anhedonia, the loss of interest in and inability to experience pleasure, is one of
the core symptoms of depression and has been shown in the CVS model (Rygula
et al., 2005; Becker et al., 2008). Increased level of anxiety has also been demon-
strated in the CVS and social defeat models (Fone and Porkess, 2008; Mineur
et al., 2006; Vidal et al., 2007; Watt et al., 2009). Depressed patients often show
cognitive dysfunctions (Austin et al., 1992), impaired visual learning and memory
has also been found in all three animal models (Fone and Porkess, 2008;
Henningsen et al., 2009; Yu et al., 2011), and impaired spatial learning can be
induced by CVS (Song et al., 2006).
Postmortem and magnetic-resonance imaging studies have revealed that the
volume of hippocampus (MacQueen et al., 2003), prefrontal cortex (Rajkowska,
218 ZOLTÁN SARNYAI ET AL.
2000), and amygdalae gray matter (Frodl et al., 2008) is decreased in depressed
patients. These morphological changes were partially reproduced in the social
isolation model (Cooke et al., 2000; Silva-Gomez et al., 2003; Day-Wilson et al.,
2006), and decreased hippocampal volume has been demonstrated in the social
defeat and CVS models (Becker et al., 2008; Jayatissa et al., 2010; Dagyte et al.,
2011). Decreased monoamine levels were observed in social isolation, social
defeat, and CVS models, mimicking some of the early human findings which
led to formulation of the monoamine hypothesis of depression (Fone and Porkess,
2008; Watt et al., 2009; Ahmad et al., 2010).
A significant portion of depressed patients show elevated circulating cortisol
(Burke et al., 2005), increased corticotrophin-releasing hormone (CRH) levels in
cerebrospinal fluid (CSF), and increased levels of BDNF in hippocampus and
prefrontal cortex (Karege et al., 2005). Many of these findings have been demon-
strated in stress-based animal models (Table II), such as elevated corticosterone
(Grippo et al., 2005; Sandstrom and Hart, 2005; Marini et al., 2006; Becker et al.,
2008) which can be normalized by antidepressant treatment in the CVS model
(Detanico et al., 2009).
Recent studies indicate that alterations in immune system activity have been
found in depressed humans, including increased serum/plasma and CSF con-
centrations of proinflammatory cytokines including interleukin-1(IL-1; Thomas
et al., 2005), IL-6 (Alesci et al., 2005), and tumor necrosis factor-alpha (TNF-a;
Mikova et al., 2001). Social isolation, social defeat, and CVS induce a proinflam-
matory effect characterized by increased TNF-a and IL-1b levels in plasma,
splenic dendritic cells, and hippocampus (Pugh et al., 1999; Wu et al., 1999;
Carobrez et al., 2002; Grippo et al., 2005; Powell et al., 2009).
One of the most widely used approaches to develop preclinical models for
psychiatric disorders is pharmacological disruption of neurotransmitter systems.
The neurotransmitter hypotheses of schizophrenia are based on mechanisms of
clinical efficacy of therapeutically effective drugs and early disease-related neuro-
chemical findings in the periphery and brain (Howes and Kapur, 2009; Coyle,
2006b). Further, administration of compounds interfering with DA and glutamate
transmission has been found to induce schizophrenia-like symptoms in nonschiz-
ophrenic humans, which could be reversed by antipsychotic medication (Javitt
and Zukin, 1991).
The amphetamine rat model mimics certain deficits of schizophrenia closely
related to positive symptoms and cognitive dysfunctions, but less so the negative
BEHAVIORAL AND MOLECULAR BIOMARKERS 219
FIG. 1. Metabolomics and proteomics changes induced by chronic treatment with haloperidol and
olanzapine in rat frontal cortex. Representative data from Ma et al. (2009) and McLoughlin et al. (2009)
showing drug-specific and common changes.
showing that olanzapine induces more metabolic side effects than haloperidol
(Deng et al., 2007). Haloperidol, however, has already been used as treatment for
Huntington’s disease (Markianos et al., 2010).
Considering the differentially expressed proteins and metabolites together, it
appears that typical and atypical antipsychotics affect brain glucose metabolism
and structural assembly of the presynaptic vesicle (Pellerin, 2003; Vaynman et al.,
2006). It is assumed that by improving neuronal activity and energy metabolism,
an amelioration of psychotic symptoms can be achieved. Antidiabetic medications
have been proposed on the basis of a potential role of altered energy metabolism
in neuropsychiatric diseases (Laron, 2009). It has been shown that intranasal
insulin improves cognition and is now a suggested therapeutic for the treatment
of Alzheimer’s disease (Benedict et al., 2011). Such reverse translational studies
using therapeutically effective drugs to identify drug-specific and class-specific
biomarkers may help to pinpoint novel disease mechanisms and drug targets.
Therefore, it may be worthwhile to consider the use of antidiabetic medication to
improve energy metabolism pathways in the brain of schizophrenia patients to
ameliorate some of the cognitive symptoms.
BEHAVIORAL AND MOLECULAR BIOMARKERS 221
IV. Statistical Methods to Link Biomarkers from Animal Models with the Human Disease
Univariate
Analysis Multivariate
B C analysis
Human Rodent
Human Rodent Human Rodent
significant significant H1 H2
analytes analytes pathway pathway H16
hits hits H2
A H3 H3
1.00 1.00 R1 R1
R2
0.75 0.75 Overlap R3 R2
R4 R5
0.50 0.50
R5
0.25 0.25 Fisher’s exact B R6 R4 R3
R7
0.00 0.00 test 12345678
R6 R7
Increased Decreased
Identification of differentially If significant potential Pathway comparison across Cluster analysis suggests Phylogenetic tree shows
expressed analytes (e.g., conclusions are species: similarity between humans that humans are more
Wilcoxon rank-sum test) - Similar pathways – Identical pathways and rodent models R1–R2 related to rodents (R1–R2
- Similar mechanisms – Different analytes as compared to R3–R7)
FIG. 2. Use of univariate and multivariate statistical methods to link biomarkers from animal models with the human disease. (A) Samples from both species
can be analyzed using methods such as LC–MSE or multiplexed immunoassay. (B) Univariate statistics can be used to detect similarities between the species.
Differentially expressed molecules in both species can be identified using the nonparametric Wilcoxon rank-sum test and Fisher’s exact test to check whether the
overlap of molecules is significant by chance. Also, pathway analysis can be used to compare the effects across the different species. (C) Multivariate approach to
detect similarity within and between the species. Hierarchical cluster analysis (HCA) groups animal models into clusters such that those within the same cluster
are assumed to be homogenous. Cluster analysis suggests similarity between three different human cohorts (H1–H3) and the two rodent models (R1–R2). A
phylogenetic tree can also be used to assess similarity. In this case, similarities between the human cohorts and R1–R2 rodent models were detected.
BEHAVIORAL AND MOLECULAR BIOMARKERS 223
In this chapter, we reviewed widely used, etiologically based animal models for
schizophrenia, MDD, and BD, and summarized key behavioral findings along
with disease-relevant alterations in neurotransmitters, molecules, cells, and cir-
cuits. What is apparent, however, is that none of these models is able to fully
replicate the human condition. This is a major problem with animal models of
psychiatric disorders and is likely to be insurmountable. Therefore, it is perhaps
advisable to move away from this traditional approach of trying to find animal
models of certain psychiatric disorders. The ongoing debate on introduction of
the new diagnostic system for mental disorders, the DSM-V, gives further impet-
uous to rethink the way we view and use animal models in this context.
Currently, the diagnosis of mental disorders is based on clinical observation
and patients’ phenomenological symptom reports (APA, 2000). It is perhaps
224 ZOLTÁN SARNYAI ET AL.
However, the most important conclusion of this exercise is that much more
research is needed to complete the RDoC matrix with behavioral and neurobio-
logical data from animal models. The RDoC domains/constructs should be
adapted to animal research, as we have attempted here. Well-validated behavioral
tests should be organized into the RDoC framework, perhaps similar to the
MATRICS initiative which has identified cognition domains that are deficient
in schizophrenia along with a ‘‘preclinical MATRICS’’ of rodent behavioral test
batteries (Young et al., 2009). Current and future animal models of psychiatric
disorders should then be tested to fill in the RDoC matrix. These RDoC matrices
can then be superimposed and overlapping patterns identified. This will lend
previously unknown translational power to the system. Emerging behavioral,
molecular, and circuit activation patterns from animal models can, therefore, be
correlated with those from human studies to provide signatures associated with
specific genetic mutations or environmental factors. Communication between the
preclinical and clinical RDoC will be needed to fulfill the promise of this ap-
proach. Integration of animal models into the RDoC framework may not only
remove psychiatric animal models from the ever-returning ‘‘validation crisis’’ but
may ultimately lead to better understanding of the pathophysiology human
neuropsychiatric diseases and to the identification of novel biomarkers and thera-
peutic targets.
Acknowledgments
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STEM CELL MODELS FOR BIOMARKER DISCOVERY
IN BRAIN DISEASE
Abstract
I. Introduction
II. Need for Cellular Models
III. Understanding Disease
IV. Biomarker Discovery
V. Accessible Cells for Biomarker Discovery in Brain Diseases
VI. Patient-Derived Stem Cells for Biomarker Discovery in Brain Diseases
VII. Olfactory Mucosa—An Accessible Neural Tissue for Biomarker Discovery
VIII. Patient-Derived Olfactory Stem Cells as Models for Brain Diseases
IX. Patient-Derived Pluripotent Stem Cells as Models for Brain Diseases
X. Advantages and Disadvantages of Current Cell Models
XI. Future Directions: Biomarkers from Stem Cell Models
Acknowledgments
References
Abstract
Most brain diseases arise from interactions between complex genetic and
environmental risk factors. Finding biomarkers for brain diseases will require
appropriate cellular models to identify dysregulated cell functions and disease-
associated biochemistries. Patient-derived stem cells hold great potential as mod-
els of brain diseases. Stem cells can proliferate and can be banked, stored, and
thawed for genomic, proteomic, and functional studies. Patient-derived, induced
pluripotent stem cells and adult stem cells from the olfactory organ in the nose are
already giving novel insights into a number of brain diseases, including Parkin-
son’s disease and schizophrenia. Biomarker discovery may be possible from
investigating disease-associated cell biologies in patient-derived stem cells.
I. Introduction
The brain is the most complex organ in the human body. The intricacies of
human evolution have resulted in unique species-specific anatomical and neuro-
biological distinctions between the human brain and the nervous systems of other
higher vertebrates. As a consequence, many brain diseases and their related
phenotypes (which we use here to refer essentially to phenotypes of interest to
clinical neurology and psychiatry) are not only complex in their nature, but
restricted to humans. This presents specific challenges for experimental scientists
interested in (1) learning more about the etiology of common human brain
disease, (2) developing biomarkers to further explore and monitor the nature of
these conditions, and (3) discovering and testing molecular interventions targeted
at modulating these phenotypes.
community; and they form a logical link between human clinical studies, postmor-
tem pathological investigations, and animal model systems. In this review, we will
provide an overview of how cellular models are being used to advance our
understanding of brain diseases, and their current and likely future roles in the
development of biomarkers and novel treatments for these conditions.
Understanding the cellular basis for many diseases is limited because of a lack
of access to living cells affected by the disease. There are relatively few biomarkers
for brain diseases that have the sensitivity and specificity to apply to individuals.
Blood cells, obtained quickly and easily, make ideal sources for biomarkers.
Lymphocytes and red blood cells are used routinely in hematology studies but
they are limited for brain diseases by their lack of obvious tissue relevance. Blood
cells are a good source of DNA for genotyping (as are cells from hair and cheek),
but they are limited in number for most cellular analyses. This limitation is often
overcome by transformation into lymphoblastoid cell lines but this genetic
STEM CELL MODELS FOR BIOMARKER DISCOVERY IN BRAIN DISEASE 243
modification also changes their phenotype, further reducing their validity. Fibro-
blasts, another commonly used patient-derived cell type, have potential because
they can be grown in large number without genetic modification. Skin fibroblasts
and transformed lymphocytes have allowed the identification of differences in
gene expression, protein expression, and cell functions in SZ and PD (Bowden
et al., 2006; Cohen et al., 1987; del Hoyo et al., 2010; Gavin et al., 2009; Hoepken
et al., 2008; Mahadik et al., 1991; Miyamae et al., 1998; Mytilineou et al., 1994;
Ramchand et al., 1994; Suzuki et al., 2008; Vawter et al., 2004; Wang et al., 2009;
Zhubi et al., 2009), although patient-control gene expression differences in SZ can
be elusive (Matigian et al., 2008). These nonneural cell models may reflect
systemic changes in cell biology associated with brain diseases (e.g., through
genetic mutation) and may provide indirect biomarkers (through the independent
mechanism defined above) associated with brain diseases, without being appro-
priate models of disease processes in the cells of the brain.
The utility of patient-derived cells diminishes with the cost of individual
biopsy and cell manipulation. Cells requiring culture and other manipulation
are likely to be less practical for individual patient assessments (compared to other
biomarkers) unless the value of the data is high enough to outweigh the high
financial and time costs. A more proximate use for patient-derived cells is their
utility as a discovery platform for novel molecular biomarkers. In this aspect,
patient-derived stem cells offer exciting prospects.
Unlike other organs, taking biopsy samples of the brain is problematic, to say
the least. Patient-derived stem cells have the potential to fill this void because they
carry the genetic makeup of the patient and can theoretically be induced to
differentiate into the cells affected in the disease (e.g., motor neurons for the
study of amyotrophic lateral sclerosis or dopaminergic neurons for examining
PD). In this context, patient-derived stem cells allow molecular investigations in
disease-relevant cell models and thus offer great potential in the hunt for novel
biomarkers (working through the casual or reactive mechanisms). Having identi-
fied such a biomarker from a cellular model, an additional challenge is encoun-
tered; there is a necessity to define biomarker levels that are sensitive and specific
enough, in a clinical context, to discriminate between individuals with or without
a particular phenotype or to meaningfully reflect the patients’ clinical status. For
example, the well-known increase in ventricular volume in SZ does not translate
to the individual because variation in the normal population reduces sensitivity
and increased ventricular volume occurs in many diseases and conditions,
244 ALAN MACKAY-SIM ET AL.
The olfactory mucosa is the organ of smell in the nose. The sense of smell is
impaired in many brain diseases, including neurodegenerative diseases (e.g.,
Alzheimer’s disease, PD) and neurodevelopmental disorders (e.g., SZ; Brewer
et al., 2003; Doty, 2009; Haehner et al., 2009; Hawkes et al., 1999). The olfactory
system is therefore sensitive to neurological disease processes, although the
mechanisms and sites of olfactory dysfunctions are not yet identified. There are
suggestions that Alzheimer’s disease and PD may arise from toxins entering the
brain through the olfactory sensory neurons in the nose (Braak et al., 2006;
Hawkes et al., 1999). These neurons are exposed to the external environment
and sensitive to toxins, leading to their destruction. Normally, the olfactory
sensory neurons are replenished through a process of neurogenesis that continues
throughout the lifetime of vertebrates, including humans (Graziadei, 1973;
Graziadei and Graziadei, 1979; Murrell et al., 1996). Neurogenesis is made
possible by ‘‘adult’’ stem cells that reside within the basal cells of the olfactory
epithelium (Chen et al., 2004; Leung et al., 2007; Mackay-Sim and Kittel, 1991).
The olfactory epithelium is the most superficial layer of the olfactory mucosa,
separated from it by a basement membrane. The stem cells of the olfactory
epithelium lie along this basement membrane and are multipotent in situ, able
to reconstitute both the neural and nonneural elements of the olfactory mucosa
(Chen et al., 2004; Leung et al., 2007). This regenerating neural tissue can be
obtained by biopsy which is easily accessible through the external naris in human
adults (Feron et al., 1998). Olfactory mucosa can be grown as organ cultures and as
dissociated cells (Feron et al., 1998; Murrell et al., 1996; Newman et al., 2000;
Wolozin et al., 1992). When grown in vitro, human olfactory stem cells are multi-
potent, able to generate neurons, and glial cells (Murrell et al., 2005; Roisen et al.,
2001) as well as many nonneural cell types of ectodermal, mesodermal, and
endodermal origin (Murrell et al., 2005, 2008, 2009).
Several brain diseases show a phenotype in the olfactory mucosa or in cells
derived from it, including Alzheimer’s disease, Rett syndrome, fragile X syn-
drome, and SZ (Abrams et al., 1999; Arnold et al., 2001; Feron et al., 1999;
STEM CELL MODELS FOR BIOMARKER DISCOVERY IN BRAIN DISEASE 245
McCurdy et al., 2006; Ronnett et al., 2003; Wolozin et al., 1993). Interestingly, no
disease-associated phenotype has been identified in primary cultures of olfactory
mucosa in PD (Witt et al., 2009). Of particular interest in the present context,
neuroblasts cultivated from patients with Alzheimer’s disease showed significant
alterations in their biochemical processing of the amyloid precursor polypeptide
(Wolozin et al., 1993) and alterations in oxidative damage (Ghanbari et al., 2004),
suggesting that this cell model is a relevant model and has the potential to identify
biomarkers for this disease. The structure of the olfactory epithelium is disrupted
in postmortem tissues from patients with SZ indicative of altered neurodevelopment
(Arnold et al., 2001), and olfactory mucosa biopsy cultures from SZ patients show
a neurodevelopmental phenotype with significantly more cells in mitosis than
occurs in cultures from healthy controls or from patients with bipolar disorder
(Feron et al., 1999; McCurdy et al., 2006). These reports demonstrate the potential
for the olfactory mucosa as a tool to investigate the biology of brain disease
phenotypes. The presence of an accessible neural stem cell in the human olfactory
mucosa has provided a new cell model for brain diseases as we recently demon-
strated for SZ and PD (Matigian et al., 2010).
Neural stem cells derived from the adult human brain or spinal cord are
grown in ‘‘neurospheres.’’ These are round, tightly packed spheroids of large
numbers of cells that form when stem cells are grown in vitro with epidermal
growth factor (EGF) and basic fibroblast growth factor (FGF2) (Rietze and
Reynolds, 2006). Neurospheres contain a small number of stem cells with their
progeny: proliferating neural precursors, and differentiating neurons and glia
(Rietze and Reynolds, 2006). Neural stem cells from adult human olfactory
mucosa are grown in similar culture conditions (Fig. 1) and, like brain neural
stem cells, have been shown to be self-renewing and multipotent, the defining
properties of stem cells (Delorme et al., 2010; Murrell et al., 2005; Roisen et al.,
2001). In our protocol, olfactory neurosphere (ONS) cells are dissociated and
grown in standard conditions as ‘‘ONS-derived’’ cells. These are adhesive cul-
tures which can be frozen, banked, thawed, and regrown in quantity for gene and
protein expression analyses and functional investigations. ONS cells can be
generated from large numbers of patients and controls, providing a platform for
multiple studies comparing patient- and control-derived cells to identify aspects of
biology that are shared by patients but different from controls (Boone et al., 2010;
Matigian et al., 2010).
246 ALAN MACKAY-SIM ET AL.
FIG. 1. Neurospheres and neurons derived from human olfactory mucosa. (A) Neurospheres, tight
spherical clusters of cells, form and detach from the underlying cells when grown in serum-free
medium containing epidermal growth factor and basic fibroblast growth factor. (B) When dissociated
and grown in differentiating medium, neurosphere-derived cells differentiate into neurons (small cell
body, long processes, immunopositive for b-tubulin III). Nuclei are stained with DAPI (blue). Bar:
30 mm in (A), 5 mm in (B).
In our initial study, we compared ONS cells from healthy controls with those
from patients with either SZ or PD. We chose to contrast these cells from patients
with two different brain diseases, both of which are complex heterogeneous
disorders: SZ, a highly heritable neuropsychiatric, neurodevelopmental disorder
(Raedler et al., 1998), and sporadic PD, a neurodegenerative disease that is
heritable only in about 5% of familial cases (Lesage and Brice, 2009). The guiding
hypothesis is that in complex, polygenic diseases, the disease mechanisms will
manifest in cell signaling pathways and genetic networks, even when single
causative genes are not present. We undertook gene expression, protein expres-
sion, pathway analysis, and cell function assays that resulted in identification
of 1700 genes and proteins which showed dysregulated expression in SZ
cells and 500 which were dysregulated in PD cells (Matigian et al., 2010). Assays
of cell function identified disease-specific reductions in glutathione levels
and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium (MTS) metabolism (a general marker of metabolic status) in PD
patient cells and increases in caspase 3 activity in SZ patient cells (Matigian et al.,
2010). Further assays of cellular function are underway to ascertain the effects of
STEM CELL MODELS FOR BIOMARKER DISCOVERY IN BRAIN DISEASE 247
Two other types of stem cells with the capacity to differentiate into neurons
are also being used as models for brain diseases. Embryonic stem (ES) cells are
pluripotent cells that can differentiate into all cell types in the body including
neurons and glial cells. Mouse ES cells were first isolated in 1981 (Evans and
Kaufman, 1981; Martin, 1981) and human ES cells in 1998 (Thomson et al.,
1998). ES cells are potential models of brain diseases caused by genetic factors
selected during preimplantation genetic diagnosis (Mateizel et al., 2006; Urbach
et al., 2004; Verlinsky et al., 2005). Induced pluripotent stem (iPS) cells are
pluripotent cells derived from differentiated cells by introducing a few key tran-
scription factor genes, originally demonstrated in adult mouse fibroblasts
(Takahashi and Yamanaka, 2006). The expression of just four transcription factors
(Oct3/4, Sox2, c-Myc, and Klf4) was sufficient to reprogram adult somatic cells back
to a pluripotent ES cell-like state. Like ES cells, iPS cells display the critical ability
to differentiate into all three germ layers, in vitro and in vivo. In 2007, the first
human iPS cells derived from fibroblasts were reported (Takahashi et al., 2007; Yu
et al., 2007). The implication was that accessible cells, such as dermal fibroblasts,
could be collected from patients, reprogrammed to an iPS cell fate, and then
redifferentiated along any lineage, so that a fibroblast could be turned into a
neuron. One of the major outcomes arising from the advent of iPS cells is that
they could be generated from patients to elucidate the cellular and molecular
bases of disease.
Although several ES cell lines have been made from embryos with known
genetic mutations, there has been little research using such cell lines to model
disease processes (Urbach et al., 2004). Perhaps, partly because of the ethical and
legal concerns about ES cells, there has been much more interest in generating
patient-derived iPS cells. Most reports have described the generation of iPS cells
from cells of patients with defined genetic forms of neurological disorders, includ-
ing from patients with familial forms of amyotrophic lateral sclerosis (Dimos et al.,
2008), muscular dystrophys, Huntington’s disease, Gaucher disease, Down’s
syndrome (Park et al., 2008), spinal muscular atrophy (Ebert et al., 2009), dysau-
tonomia (Lee et al., 2009), SZ (Brennand et al., 2011; Chiang et al., 2011) and PD
(Nguyen et al., 2011; Park et al., 2008). These investigations demonstrate proof-of-
principle that iPS cells can be derived from patients, that these iPS cells can
differentiate into neurons, and that they demonstrate altered expression of the
genes and proteins of interest. As such, these publications are forerunners of an
approach which will be useful for defining the molecular and cellular conse-
quences of genetic mutations in neurons.
iPS cells have also been isolated from patients with neurological disorders of
undefined genetic and/or environmental contribution (Brennand et al., 2011;
STEM CELL MODELS FOR BIOMARKER DISCOVERY IN BRAIN DISEASE 249
Soldner et al., 2009). These studies now demonstrate the feasibility of making iPS
cells and, from these, neurons. While iPS cells have the potential to be generated
from patients with any neurological diseases, they have not yet revealed the
molecular and/or cellular basis of complex genetic and/or environmental inter-
actions that lead to diseases such as Alzheimer’s disease, PD, or SZ. The success of
genetic investigations in identifying monogenetic forms of neurological diseases
such as those mentioned above has provided critical mechanistic insights into the
pathogenic pathways that likely underpin sporadic forms. However, it is impor-
tant to recognize that these genetically loaded cases account for a minority of the
disease burden. For example, the most common form of genetic PD (caused by
LRRK2 mutations) occurs in < 1% of PD patients in Australia (Huang et al., 2007).
Likewise, SOD1 mutations account for less than 1% of all amyotrophic lateral
sclerosis cases and it is sobering to reflect on the fact that lead molecules emerging
from large drug discovery efforts based on the outcomes of SOD1 animal studies
have proven to be ostensibly unsuccessful in human amyotrophic lateral sclerosis
(see Schnabel, 2008). In contrast, a major aspect of cellular models of neurological
disorders is their capacity to represent, or at least account for, the wide genetic
backgrounds against which most cases of the disease arise. In this aspect, iPS cells
have been made from five patients with sporadic PD, from which neurons were
generated (Soldner et al., 2009). Interestingly, these authors did not find pheno-
typic differences between sporadic PD iPS and control iPS cells despite the fact
that these iPS cells were derived from LRRK2 mutation carriers (Nguyen et al.,
2011). In another disease of complex genetics, SZ, iPS cells have been made from
two patients with a DISC1 mutation (a familial monogenic form; Chiang et al.,
2011) and three patients with familial SZ of unknown genetics (Brennand et al.,
2011). This latter study identified reduced neurite branching and diminished
neuronal connectivity and reduced glutamate receptor expression in neurons
generated from iPS cells. This demonstrated that iPS cells can be useful for
identifying disease-associated changes in cell biology in neurons, an apposite
proof-of-principle for a cell model of a brain disease of unknown genetics.
At present, there are two primary limitations to the use of iPS cells as models
of sporadic cases of neurological disorders. The first is technical. The efficiency of
generating iPS cells still remains low (Hanna et al., 2009). Therefore, most
published studies are based on from 1 to 5 iPS clones. Given the clonal variation
of iPS cells (Laurent et al., 2011) derived from a single source of primary cells, it is
not clear precisely what data from a single patient clone compared with a single
healthy control clone represents. At present, the generation and expansion of iPS
cells is not a trivial exercise, although advances in technology should overcome
many present bottlenecks. Yet for complex diseases such as idiopathic PD and
sporadic SZ, it might be necessary to compare hundreds of patient and control
cell lines to gain insight into molecular mechanisms. Another limitation is not so
much technical as inherent in the iPS derivation process. This limitation is the
250 ALAN MACKAY-SIM ET AL.
This field of study is rapidly advancing and it is now possible to convert fibroblasts
directly into neurons without passing through a pluripotent stage (Vierbuchen
et al., 2010).
It is still early days for cell models of brain diseases. The utility of current cell
models varies with advantages and disadvantages associated with each cell type
(Table I). For example, patient-derived cells vary in the ease with which they can
be obtained and generated. Blood lymphocytes are easy to obtain but cannot be
propagated without transformation into lymphoblastoid cell lines. Fibroblasts
require a more invasive biopsy procedure but can be grown in vitro and banked.
Biopsying the olfactory mucosa is still relatively more invasive but ONS cells are
similar to fibroblasts in the time taken to generate these in vitro and are similarly
robust for cell culture and banking. iPS cells are labor intensive to generate and
maintain compared to other patient-derived cells, requiring many months, rather
than weeks, to generate and additional months for validation. They are also more
temperamental to maintain in vitro.
Cell models also vary in their relevance as representatives for neural tissues
and their potential for reflecting neural biology. For example, ONS cells (derived
from a neural tissue) but not fibroblasts (derived from skin) show gene expression
differences in neurodevelopmental signaling pathways in SZ (Matigian et al.,
2010). However, fibroblasts can show disease-associated differences that may be
associated with less tissue-specific, but systemic, changes such as cell proliferation
(Wang et al., 2009) and adhesion (Mahadik et al., 1994). Both ONS cells and iPS
cells can be differentiated into neurons but iPS cell technology is more advanced
in terms of reliability and for rapid production of neurons of different classes.
A potential disadvantage of iPS cells is the unknown and unpredictable effect of
Table I
UTILITY OF PATIENT-DERIVED CELLS AS DISEASE MODELS.
Potential
Adult Easy for
patient- Easy to to Genetically Proliferate longitudinal Differentiate
derived obtain grow modified in vitro monitoring into neurons
Nonstem cells
Lymphocytes ✓ ✓ ✓ ✓
Lymphoblastoid ✓ ✓ ✓ ✓ ✓
cells
Fibroblasts ✓ ✓ ✓ ✓ ✓
Stem cells
ES cells ✓ ✓
iPS cells ✓ ✓ ✓ ✓
ONS cells ✓ ✓ ✓ ✓ ✓ ✓
252 ALAN MACKAY-SIM ET AL.
For the first time for many diseases, stem cell science will provide the ability to
investigate the cellular and molecular basis of brain diseases in living neural cells
from patients, including neural progenitor cells, neurons, astrocytes, and oligoden-
drocytes. These cell models will carry disease phenotypes, genetics, and epige-
netics, and span the variability encountered across the patient population. They
also provide the potential to identify novel molecular markers that distinguish
patient from control cells. These may be altered levels of gene or protein expres-
sion, or altered levels of metabolites. The cellular models may also provide the keys
to differential diagnosis or indicators of progress of a disease or its treatment.
The ideal next steps would be to source these markers in blood or other easily
accessible tissues such as skin, hair, or cheek cells. It may be necessary to sample
from more ‘‘neural’’ sources such as cerebrospinal fluid or olfactory mucosa. It
may also be possible in the not-too-distant future that molecular biomarkers could
be monitored in vivo using the rapidly advancing brain imaging techniques such as
magnetic resonance spectroscopy (Soares and Law, 2009).
Acknowledgments
This work was supported by funding to the National Centre for Adult Stem
Cell Research from the Australian Government Department of Health and Aging.
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THE APPLICATION OF MULTIPLEXED ASSAY SYSTEMS FOR
MOLECULAR DIAGNOSTICS
Emanuel Schwarz1, Nico J.M. VanBeveren2, Paul C. Guest1, Rauf Izmailov3 and
Sabine Bahn1,4
1
Department of Chemical Engineering and Biotechnology, University of Cambridge,
Cambridge, United Kingdom
2
Department of Psychiatry, Erasmus University, Medical Centre, Rotterdam,
The Netherlands
3
Rules-Based Medicine, Inc., Austin, Texas, USA
4
Department of Neuroscience, Erasmus Medical Centre, Rotterdam, The Netherlands
Abstract
I. Introduction
II. The Problem of Disease Heterogeneity
A. History of Clinical Diagnosis of Psychiatric Disorders
B. Advantages and Disadvantages of DSM Based Diagnosis
C. How Do These Issues Affect Biomarker Research?
III. Multiplexed Assays are Needed to Characterize Heterogeneous Illnesses
IV. Multiplex Immunoassay Profiling
V. Toward Functional Analysis
VI. Conclusion and Outlook
Acknowledgments
References
Abstract
I. Introduction
The search for biological markers of psychiatric disorders that have applica-
bility as clinical tools has been ongoing for several decades. There is now signifi-
cant interest in the discovery of such markers, as they could be useful as objective
tools to assist the diagnosis, treatment selection, and monitoring of patients.
Biomarker readouts reflecting abnormal molecular processes such as the niacin
skin flush response test have shown early promise for application as a schizophre-
nia diagnostic (Horrobin, 1980). This test is based on an attenuated flush response
of schizophrenia patients after topical application of niacin to the skin. Ultimately,
the low sensitivity and specificity, along with the differences between acute and
multiepisode schizophrenia subjects, have prevented clinical introduction of this
method (Ward et al., 1998; Puri et al., 2001; Smesny et al., 2005).
Molecular research into psychiatric disorders such as schizophrenia, bipolar
disorder, or major depressive disorder has shown that, despite the high degree of
heritability, single genes only confer a small proportion of the overall risk of onset
(Schwarz and Bahn, 2008). Also, studies targeted against single molecules have
met with less than promising results. This is most likely due to the fact that
molecules measured in peripheral body fluids or tissues often show only subtle
changes in molecular levels which result in a small effect size. This is typically not
sufficient for discriminating patients from healthy volunteers, or from patients
with other disorders, with suitable performance. One way around this problem is
to use large sample numbers for identification of statistically significant abnorm-
alities. However, this is often challenging due to the limited availability of well-
characterized high-quality samples which have been collected according to
uniform standard operating procedures. In particular, the treatment state and
disease stage of the subjects in question can pose potential confounding factors in
molecular and other empirical investigations. For this reason, it may be most
relevant to study individuals at the earliest stages of the disease before any
treatment has been applied and before the disease has progressed. This is a
challenging task as even large clinical centers generally collect samples from less
than 30 drug-naive first-episode schizophrenia patients per year. Such confound-
ing factors are also a particular problem for cross-disease comparisons which are
essential in the development of differential diagnostic tests.
Emerging proteomic and transcriptomic platforms have facilitated the simulta-
neous measurement of hundreds or thousands of molecules, enabling nonhypothesis
driven profiling approaches. Such profiling methods have been performed for
various psychiatric disorders (Lakhan, 2006), but the resulting molecular candidates
have not been validated or translated on a larger scale. Our group has recently
employed an approach based on multiplexed immunoassay profiling which resulted
in identification of a serum signature that could identify schizophrenia patients with
THE APPLICATION OF MULTIPLEXED ASSAY SYSTEMS 261
an accuracy of 82% across five independent patient cohorts (Schwarz et al., 2010a).
Along with some components of this molecular signature that showed reproducibili-
ty across independent clinical sites, many of the molecules were found to be changed
only in specific centers. The most likely interpretation of this effect is that, this is a
molecular reflection of heterogeneity in a population of patients in different centers,
despite the fact that these appeared to have comparable clinical presentations.
Therefore, the use of multiplexed immunoassays was necessary to achieve this
performance, given the greater discriminatory power afforded by multiple assays.
The combined use of multiple molecular measurements can lead to greater sensitiv-
ity and specificity in the same way that a complete fingerprint aids forensic scientists
in the absolute identification of an individual, whereas a partial fingerprint leads to a
higher degree of uncertainty.
These issues give rise to the question of how far the construct validity of
psychiatric diagnoses impacts on biomarker discovery. Currently, psychiatric dis-
orders are diagnosed based on subjective interviews and patient history. Inevita-
bly, the question arises of how molecular data derived from subjectively diagnosed
patients can lead to the development of objective molecular diagnostics. In this
chapter, we will discuss the challenge of performing molecular biomarker discov-
ery in the case of subjects with schizophrenia and other psychiatric illnesses. The
review is organized into four parts. The first part addresses the current practice
and challenges of the clinical diagnosis of psychiatric disorders. The aim of this
section is to illustrate the concept of diagnostic validity for psychiatric disorders
and the potential impact of this on biomarker research. The second part will
discuss the associated challenges and methods for the investigation of biomarkers.
In particular, we will elaborate on two main types of approaches to identify
disease-related molecular abnormalities when there is uncertainty regarding the
validity of the clinical diagnosis. The third part of the review will present an
introduction to the multiplex immunoassay approach that facilitates identification
and quantitation of molecular biomarkers, and the fourth part will cover novel
approaches for extending these molecular findings into the realms of identifying
the associated functional consequences.
(Sadler, 2004), with the 1994 DSM-IV as the most recent edition (American
Psychiatric Association, 1994), and the revised DSM-V expected in 2012.
While the influential DSM-III was introduced in 1980, the fundamental
structure of this system dates back to the late nineteenth-and early twentieth-
century, when Kraepelin made his influential distinction between dementia prae-
cox and mania (Kraepelin, 1971). Kraepelins’ distinction stemmed from the
observation that, among the general group of relatively young previously normal
functioning patients who developed alterations in behavior and mental abilities,
two distinct long-term patterns could be identified. The most serious cases were
those individuals who failed to show complete recovery, exhibiting lasting deficits
throughout life. It was for this group that the term dementia praecox was initially
used. The other group was comprised those patients who clearly improved to a
state of almost complete recovery, although a remitting-relapsing course could
arise. This latter group formed the manias. That such a distinction could be made
with any validity has been fundamental to psychiatric classificatory systems since
this time.
Dementia praecox later developed into the schizophrenia concept (Bleuler
and Zinkin, 1950), whereas mania formed the basis of manic depression and
depressive disorder. Finally, the distinction was incorporated into the DSM-III
system, with the group of psychotic disorders incorporating schizophrenia on one
side, and the broad group of mood disorders incorporating manic depression
(bipolar disorder) and depressive disorders (major depressive disorder) on the
other side (for an overview of the work of Kraepelin and the influence on
psychiatric classification, see Braceland, 1957; Decker, 2004).
Apart from mood and psychotic disorders, three other major diagnostic
categories were incorporated into the DSM system. The work of Kanner and
Asperger on young children with odd, self-oriented behavior, and inability to
engage in normal reciprocal social contacts gave rise to the broad group of autism
spectrum disorders (Asperger, 1944; Kanner, 1968). Also, a group of anxiety
disorders was recognized, partly because of the many traumatized veterans
from the major twentieth century military conflicts, incorporating posttraumatic
stress disorder and panic disorder (Bienvenu et al., 2010). In addition, personality
disorders emerged from Freud’s neurosis concept (Reed, 1990).
The research scientists and clinicians responsible for developing DSM-III in
the late 1970s were conscious of the fact that little was known about the biological
underpinnings of psychiatric disorders. At the time, a psychological explanation
existed for most disorders, usually in the form of some kind of stress-reaction
process (van Praag, 1997). However, it was agreed upon that the scientific basis for
psychological explanations was limited. Therefore, the DSM-III and DSM-IV
systems do not take into account any hypothesized cause or process which may
underlie the diagnostic categories, in order to avoid the possibility that etiological
preconceptions would interfere with subsequent research (American Psychiatric
THE APPLICATION OF MULTIPLEXED ASSAY SYSTEMS 263
Since their introduction, DSM-III and DSM-IV have been praised for initiat-
ing a rigorous, methodologically sound approach to psychiatric diagnostics, im-
proving the quality and quantity of psychiatric research. Indeed, the burgeoning
field of psychiatric research as it is today would most likely not exist without the
DSM system. Over the years, however, the DSM approach has also met with
severe criticism (van Praag, 1997, 2000, 2001; Sadler, 2004). It has been argued
that research into the biological determinants of abnormal behavior exacts
particular standards upon psychiatric diagnosis and that the DSM system falls
short in several respects. Clearly, diagnosis is the principal rate-limiting step in
biological psychiatric research, and when researchers use invalid diagnostic cate-
gories, the results of the research will be either absent, clouded by inclusion noise,
spurious, or, in the worst case, misleading.
The main disadvantage of the DSM diagnostic categories is that they do not
reflect a true medical diagnosis, but they are arbitrary categories to a certain
extent (van Praag, 1997). Through training of clinicians and psychiatrists in the
264 EMANUEL SCHWARZ ET AL.
use of standardized interviews, the use of DSM can lead to acceptable interrater
validity. However, such increased reliability does not necessarily imply validity of
the identified constructs. It has repeatedly been argued that the validity of DSM
constructs is limited with respect to the underlying pathophysiological pathways,
delimitation from other disorders, and in follow-up studies.
Specifically, DSM categories are notoriously heterogeneous since the system
allows for several combinations of symptoms to be arranged into one category. For
example, the schizophrenia concept can be generated out of 23 different combi-
nations of symptoms and phenomena. This heterogeneity is also reflected in the
debate about the validity of certain diagnostic groups within the schizophrenia
spectrum such as in the case of schizoaffective disorder (Maj et al., 2000).
A specific development of the past years has also contributed to the heterogeneity
found in DSM-IV categories. Some researchers argue that there exists a continuum
between core psychiatric symptoms and syndromes, and normal functioning (van Os
et al., 2000). This has lead to a situation in which the border between mental distress
and mental illness is only vaguely marked. van Praag likened this situation to
searching for the pathogenesis of tuberculosis but not to making a diagnostic
distinction between this and the common cold (van Praag, 1997). He found this
issue specifically prominent in research of depressive disorders, in which the distinc-
tion between sorrow and depression is not adequately made.
Taken together, these issues have led to the need for elucidation of the
biological mechanisms of discrete disorders in biological psychiatry, including
through the search for molecular biomarkers. However, one may question the
construct validity of these distinguished disorders, as many of these seem to
represent a variety of more or less comparable but, in many ways, dissimilar
conditions. As a result, it is hard to believe that the search for particular brain
dysfunctions underlying such heterogeneous diagnostic constructs stands much
chance of success.
the approach our group has taken in identifying a biomarker profile for schizo-
phrenia, and investigating patients with atypical symptoms who later developed
schizophrenia (Schwarz et al., 2010a). This inherently assumes a high level of
resolution and the presence of individual symptoms associated with a specific
molecular phenotype. It remains to be determined whether this assumption
reflects the biological reality.
The second method works the other way round and might thus be called ‘‘the
broad-to-narrow’’ approach. This approach assumes that current diagnostic
categories have limited value with respect to their underlying biological validity,
whether determined by DSM, historically defined archetypical syndromes or by
dimensionally defined psychological functions and should therefore not be used to
steer biomarker research. Instead, the starting point should be the clinical reality
that patients come into broad ‘‘problem basins’’ (van Praag, 2000), such as the
mental and behavior disorders seen in young children, adolescents, or adults. In
this approach, a first step would be to collect a large group of patients from one of
the problem basins, such as adolescent-onset mental and behavioral disorders.
Such a group is likely to comprise the current DSM diagnoses psychotic disorders
including schizophrenia, bipolar disorder, major depressive disorder, conduct
disorder, and various developing personality disorders. The next step would be
to measure a multiplex biomarker fingerprint (i.e., a serum proteome profile),
assuming that alterations in serum proteins will be present even though the exact
nature or causes of these changes is unknown. After this, computerized clustering
techniques could be used to identify clusters of patients with similar biomarker
profiles. Patients belonging to a single cluster will, by definition, have the same
biological profile, at least with respect to the biomarkers and tissue investigated.
Such a cluster might be associated with a patient exhibiting the specific signs and
symptoms of a traditional diagnostic category. However, it is more likely that a
cluster will consist of mixture of patients exhibiting the signs and symptoms of a
variety of DSM disorders. Therefore, it should be investigated whether the
patients of a single cluster show a common, meaningful clinical characteristic
such as a similar prognosis, developmental trajectory, response to treatment, or
alterations in biological or psychological function.
This approach overcomes the above-mentioned assumption that symptoms
have to be associated with specific molecular underpinnings. In fact, patient
clustering based on molecular data alone may yield a partitioning of psychiatric
patients that does not align with the current diagnostic criteria. In this way, patient
clustering could ultimately lead to changes in current diagnostic criteria, in
particular if patients of distinct clusters show different treatment response or
side effects. The main difficulty with this approach is the requirement of large
numbers of samples that are comparable from a purely analytical point of view.
Such comparability requires the implementation and adherence to strict standard
operating procedures for sample collection across multiple clinical sites. Also the
THE APPLICATION OF MULTIPLEXED ASSAY SYSTEMS 267
Due to the wide spectrum of symptoms, it is likely that the molecular basis of
the current classification of schizophrenia and other psychiatric disorders is not
reflected in single, but in multiple molecular alterations which might differ in their
importance between individuals. This is supported by several different molecular
investigations which found only weak contributions of single genes to the risk of
schizophrenia onset, despite the high heritability of the disorder. Molecular
alterations found in more accessible, peripheral body fluids are typically low
due to the large biological variability observed in asymptomatic individuals. As
mentioned above, this will lead to low effect sizes, necessitating the use of higher
sample numbers and, consequently, a larger number of molecular assays to obtain
sufficiently high diagnostic accuracy. In our study, we identified a panel of 51
molecular assays which, when tested in a larger population, yielded a sensitivity
and specificity of 82% for discriminating schizophrenia patients from controls
(Schwarz et al., 2010a). As stated above, the applied approach was based on
multiplexed immunoassay technology that measures a prespecified set of analytes
in every sample. This approach could be considered as ‘‘semihypothesis’’ driven
since the measured molecules are derived from pathways that have high relevance
for the disorder under investigation although no prior hypothesis about the
alteration of individual molecules exists. Also, this method allows the measure-
ment of candidate biomarkers that have been implicated in the scientific literature
or have been found to be changed by other investigators. The measurement of
molecules in a multiplex immunoassay system has the advantage that individual
molecules with an effect size that is too low to lead to a biomarker may become
useful when it is combined with other molecules.
As described above, the combination of multiple weak classifiers can lead to a
classification rule with high performance (Freund and Schapire, 1997). Short-
comings of this method are the high cost of incorporating newly discovered
molecules as novel assays into the multiplexed system and the absolute reliance
on the availability suitable specific antibodies in order to achieve this. In particu-
lar, the latter requires a careful experimental setup and adherence to stringent
quality control procedures to avoid shifts in measurement performance caused by
changes in reagent batches. Such shifts can be detrimental for the application of a
multiplexed system as a diagnostic tool. Decision rules that produce a combined
output based on the measurement of the multiplexed molecules need to be trained
on a reference set of samples. This inherently requires that all future samples
should be comparable in their measurement relative to this reference set. This
problem is exacerbated as more molecules become required for the decision rule
since analytical deviations of individual measurements may add up, such that the
combined classifier produces a noisy output.
THE APPLICATION OF MULTIPLEXED ASSAY SYSTEMS 269
TM
This section describes the procedure of the mutiplex-analyte profiling (MAP )
platform for profiling serum samples taken from patients with psychiatric disorders.
The recent development and application of such multiplex immunoassay platforms
allows the simultaneous measurement of tens to hundreds of molecules in individual
samples. The Rules-Based Medicine (Austin, TX, USA) MAP technology has
already been applied successfully in numerous clinical studies targeting diseases
such as epithelial ovarian cancer (Bertenshaw et al., 2008), scleroderma (Duan et al.,
2008), coronary artery disease (Gurbel et al., 2008), myocardial infarction (Escobar
and Lindsey, 2007), autoimmune disorders (Delaleu et al., 2008), and sickle cell
anemia (Lee et al., 2007). This platform is also suitable for the development of
sensitive and specific tests for use in medical practice.
The assays are basically a combination of immunoassay (reviewed elsewhere:
Lowry et al., 1989) and flow cytometry (reviewed elsewhere: Norman, 1980)
techniques. A typical assay begins when a small volume from each sample is
added to reaction wells in a plate containing the capture microspheres (Fig. 1).
The microspheres are typically conjugated to antibodies and are encoded with a
unique fluorescent signature that is specific to the targeted molecule. The antibody-
microsphere conjugates are incubated with the samples to allow the molecules of
interest sufficient time for binding. After this, a cocktail of specific, biotinylated
detection reagents is added, followed by the addition of a fluorescent reporter
molecule which is usually another antibody which recognizes a different epitope
on the molecule of interest. Finally, the mixture is washed to remove unbound
TM
detection reagents prior to reading the reaction plate in the Luminex machine.
The Luminex instrument operates similar to a flow cytometer, using the princi-
ple of hydrodynamic focusing to channel the microspheres containing bound
270 EMANUEL SCHWARZ ET AL.
Antibody-microsphere
conjugate
Ide
ntit
y
ity
ant
Qu
molecules and assay reagents, one at a time, along a path that is analyzed by two
lasers (Fig. 2). The excitation beams of the red laser measures the unique fluorescent
signature of each microsphere, and the green laser determines the amount of
fluorescence generated in proportion to the concentration of the molecule in the
sample. Data are acquired and reported in real time, affording the ability to
repeatedly measure the concentration of a given molecule in each sample.
The MAP tests incorporate specific controls for each molecule assayed within
the multiplex. These controls also mimic the sample matrix, creating a realistic
background for the measurements. For the majority of tests, native proteins are
used as controls rather than recombinant proteins. The benefits of native controls
include better assay performance over time and a greater specificity in measuring
the native counterparts within the samples. In addition, low, medium, and high
concentrations of controls are used to support data accuracy across the concentra-
tion range which has already been defined by testing of standards. The control
THE APPLICATION OF MULTIPLEXED ASSAY SYSTEMS 271
values are also used to monitor assay performance longitudinally. In most cases, the
intraassay coefficient of variation is less than 10%. The method also uses a set of
modified Westgard rules (Westgard, 1992) (http://www.westgard.com/index.php)
to evaluate the control data. This includes a set of multirule quality control decision
criteria that are used to determine whether or not an assay functions as expected.
This raises alerts to potential problems such as anomalies in individual control
value levels, systematic problems among or within the controls, or potential
unwanted trends in the data. The controls must pass all of quality control criteria
to be considered valid, and only results that meet this standard are reported.
The final step in the process is the calculation and reporting of the experimen-
tally determined molecular levels in the samples. As with most immunoassay based
systems, this is achieved by plotting the readings of each sample along standard
curves to derive the concentration of the target molecule. The corresponding
concentration determined for that sample can then be adjusted by the appropriate
dilution factor to calculate the absolute concentration of the molecule of interest.
In terms of further quality control measures, one of the benefits of multiplexing is
that proteins that are likely to be present in a particular sample type can be used as
a point of reference. Thus, the absence of a robust signal from such a reference
marker may indicate reagent or liquid handling issues.
We have recently applied this methodology in collaboration with Rules-based
medicine in studies of schizophrenia (Schwarz et al., 2010a), bipolar disorder
(Herberth et al., 2011), and Asperger syndrome (Schwarz et al., 2010b). In each
case, a robust multiplex signature was capable of distinguishing patients from
controls with good precision. Also, these studies led to an increased biological
understanding of these conditions at the molecular level. In the case of the bipolar
disorder study, the identified molecules were associated with cell survival path-
ways (Herberth et al., 2011), and the Asperger syndrome study revealed sex-
specific alterations in immune pathways in males and in growth factor and
hormonal systems in females (Schwarz et al., 2010b). In studies of schizophrenia,
we found alterations in the levels of insulin and some hormones of the hypotha-
lamic-pitutary-adrenal-gonadal axis which is consistent with reports of insulin
resistance in at least some of these subjects (Guest et al., 2010, 2011; Spelman et al.,
2007; see chapter ‘‘Abnormalities in endocrine and metabolic function in psychi-
atric disorders’’ by Guest et al.).
Up to this point, the assays highlighted in this chapter have described the
multiplex assay format for measurement of the levels of proteins and small
molecules in body fluids and tissues. However, this information does not
272 EMANUEL SCHWARZ ET AL.
B
K A + B = intact proinsulin
R
A + C = total proinsulin
S S
R
S S R
S C
total proinsulin
S - intact proisulin
des 31,32 proinsulin
FIG. 3. Two-site immunoassays for measurement of intact proinsulin and des 31,32 proinsulin. Use
of antibody A for capture and antibody B for capture recognizes intact proinsulin as this requires an
intact cleave site at amino acids 31 and 32 (RR). Use of antibody A for capture and antibody C for
detection recognizes all forms of proinsulin with a continuous C-peptide and will therefore recognize
intact proinsulin, des 31,32 proinsulin, and des 64,65 proinsulin (total proinsulin). The levels of des
31,32 proinsulin can be calculated by subtraction of the levels obtained using antibodies A and B (intact
proinsulin) from that with antibodies A and C (total proinsulin). The levels of des 64,65 (KR) proinsulin
is discounted as these have been found to be negligible in vivo cleavage at amino acids 64 and 65 (KR)
(Guest et al., 2010).
et al., 1989). A similar format has been used to detect changes in glycosylation of
transferrin, in this case, using an antibody against the transferrin peptide back-
bone and a lectin molecule which recognizes galactose residues (Pekelharing et al.,
1987). Likewise combinations of antibodies which target the peptide backbone
and specific phosphorylated amino acid residues have been used to measure
phosphorylation and dephosphorylation of proteins, a key regulatory feature in
cellular signal transduction. One example of such an assay is the demonstration of
changes in phosphorylation of tyrosine 1248 in the erythroblastic leukemia viral
oncogene homolog 2 (ERBB2) breast cancer-related protein (Cicenas et al., 2006).
The general format for a phosphorylation-based immunoassay is shown below
TM
using the MAP bead technology (Fig. 4).
Studies of glycosylation and phosphorylation changes may be important in
investigations of psychiatric and neurological disorders. Recent studies have
identified N-linked glycosylation changes in the cerebrospinal fluid and serum
in patients with schizophrenia (Stanta et al., 2010). Reduced phosphorylation of
the NMDA receptor NR1 subunit has also been observed in postmortem brain
tissue from schizophrenia subjects (Emamian et al., 2004). Therefore, translation
of assays which can assess the states of such posttranslational modifications to the
TM
MAP format would be a benefit in future studies of psychiatric conditions. The
use of multiplex assays in this case would lead to increased accuracy and reliability
274 EMANUEL SCHWARZ ET AL.
P P P P
P P
P P
Add sample containing Add labelled
phoshorylated proteins antibody specific for
phosphorylation
Acknowledgments
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ALGORITHM DEVELOPMENT FOR DIAGNOSTIC
BIOMARKER ASSAYS
Abstract
I. Introduction
II. Methods
A. Study Participants
B. Serum Samples
C. DiscoveryMAP Multiplex Immunoassay Profiling
D. Biomarker Selection
E. Multiplex Assay Construction
F. Decision Rule Development
III. Results and Discussion
A. Schizophrenia Biomarker Selection
B. Decision Rule Optimization
C. Decision Rule Performance
D. Decision Rule Refinement
E. Decision Rule Recalibration
IV. Conclusions
Acknowledgments
References
Abstract
changes associated with different reagent lots. The resulting decision rule deliv-
ered a sensitive and specific prediction for presence of schizophrenia in subjects
compared to matched controls, with a receiver operating characteristic area
under the curve of 88%. Performance of the recalibrated decision rule remained
constant across lot changes, ensuring consistency and accuracy.
I. Introduction
II. Methods
The present study consisted of three phases. The first phase was aimed at
selection of accurate and reproducible schizophrenia biomarkers from a collection
of 181 molecular assays within the Rules-Based Medicine DiscoveryMAP assay
collection. Phase 1 resulted in the selection of 51 specific immunoassays to be used
in assay validation. Phase 2 featured a refinement of the individual components of
the multiplexed immunoassay, development of a decision rule for separating
schizophrenia patients from normal controls, and validation of the decision rule
using a cohort of 806 clinical samples. For biological validation of the decision
rule, 480 of these samples were analyzed only during phase 2 of this study. The
protocols for the study participants, clinical samples, and test methods were
carried out in compliance with the Standards for Reporting of Diagnostic Accu-
racy (STARD) initiative (Bossuyt et al., 2003). Phase 3 addressed the implementa-
tion practice of the constructed decision rule, which had to deal with variability of
molecular measurements across different lots of reagents. As a result, the decision
rule was further refined and recalibrated using the 40 most robust assays from the
original panel of 51. In addition, a general recalibration procedure yielding only
limited changes of the offset coefficient was introduced.
A. STUDY PARTICIPANTS
The subjects were recruited from the Departments of Psychiatry at the Uni-
versities of Cologne (cohort 1), Münster (cohort 2), Magdeburg (cohorts 3 and 4),
Rotterdam (cohort 5), and the USA military (n ¼ 110 bipolar disorder (BD)
282 RAUF IZMAILOV ET AL.
patients and n ¼ 110 healthy controls). Cohorts used for the molecular assay
selection phase were comprised of 250 first- and recent-onset schizophrenia
patients and 230 healthy control subjects (Table I). Schizophrenia patients of
cohort 1 (n ¼ 71), 2 (n ¼ 46), 4 (n ¼ 47), and 5 (n ¼ 40) were first onset and
antipsychotic-naı̈ve, and 32 of 46 subjects from cohort 3 had not been treated with
antipsychotic medication for more than 6 weeks prior to sample collection. First
onset antipsychotic-naı̈ve patients are difficult to recruit since even large clinical
facilities can only expect to diagnose about 20–30 such patients each year. To
facilitate the future development of a test with differential diagnosis capability, we
also carried out DiscoveryMAP analysis using samples from subjects within
30 days before their first contact with USA military psychiatric services and
who later received a confirmed diagnosis of BD (n ¼ 110, Table II). The cohort
used to validate and implement the decision rule comprised samples from a
mixture of first onset and chronic antipsychotic-treated schizophrenia (n ¼ 577)
Table I
DEMOGRAPHIC DETAILS OF SUBJECTS INCLUDED IN BIOMARKER SELECTION PHASE.
Class Cohort 1 2 3 4 5
Control n 59 46 45 40 40
M/F 31/28 35/11 27/18 33/07 26/14
Agea 30 8 27 9 34 12 27 4 36 11
BMIa 23 4 na 24 4 na 24 3
Schizophrenia first onset n 71 46 46 47 40
M/F 42/29 35/11 30/16 36/11 27/13
Agea 31 10 27 9 35 12 26 8 35 10
BMIa 24 5 22 2 26 5 na 25 5
a
Values are shown as mean SD.
Table II
DEMOGRAPHIC DETAILS OF PRESYMPTOMATIC BIPOLAR DISORDER AND CONTROL SUBJECTS.
Control n 110
M/F 70/40
Agea 21 4
Presymptomatic bipolar n 110
M/F 70/40
Agea 21 4
a
Values are shown as mean SD.
ALGORITHM DEVELOPMENT FOR DIAGNOSTIC BIOMARKER ASSAYS 283
Table III
DEMOGRAPHIC DETAILS OF 707 SUBJECTS (51-PLEX VALIDATION).
Class Cohort 1 2 3
Control n 72 84 73
M/F 31/40a 41/43 51/22
Agea 31 9 37 14 34 11
BMIa 24 3 na 25 4
Schizophrenia first onset
Drug naı̈ve n 132 18 56
M/F 78/54 14/4 36/20
Agea 30 9 28 9 37 11
BMIa 23 4 22 3 25 5
Treated n 130 71
M/F 73/56a 49/22
Agea 34 12 26 8
BMIa 25 5 24 4
Schizophrenia chronic
Drug free n 11
M/F 8/3
Agea 32 9
BMIa 26 6
Treated n 60
M/F 32/28
Agea 33 9
BMIa 26 5
99 follow-up samples were available from patients in cohort 2, yielding a total sample number of 806.
a
Demographic information for one patient not available.
284 RAUF IZMAILOV ET AL.
patients and respective controls (n ¼ 110) were selected from a USA military
serum bank comprising approximately 43 million sera, which facilitated matching
for age, gender, ethnicity, and lifestyle.
B. SERUM SAMPLES
D. BIOMARKER SELECTION
After the new multiplexed immunoassays were developed, these were used to
measure all 51 molecules on a combined data set of 806 subjects comprising 577
schizophrenia and 229 control subjects (from three cohorts: Universities of
Cologne, Münster, and Magdeburg, Germany). The resulting 51 806 matrix
of data was used to develop a decision rule for separating schizophrenia patients
from healthy controls. There are a variety of ways of designing such decision rules.
In this study, we used one of the most reliable approaches, SVM (a general
description of SVM theory and its application can be found in Vapnik, 1998).
Since its introduction in early 1990s, SVM technology became the default choice
for classification algorithms. In particular, SVM is capable constructing nonlinear
decision rules in multidimensional input spaces by using a so-called kernel
function. However, in order to keep the decision rule simple in implementation,
we opted for linear SVM. In other words, the kernel function is linear, and the
decision rule created a separating linear hyperplane in the corresponding input
286 RAUF IZMAILOV ET AL.
In this section, using the methods described in the previous section, we report
the results of biomarker selection and development of classification decision rule.
The new multiplexed immunoassays were used to analyze sera from 806
subjects comprising 577 schizophrenia patients and 229 controls (from three
cohorts: Universities of Cologne, Münster, and Magdeburg). The resulting 806
vectors of dimension 51 were used to develop a linear decision rule for separating
schizophrenia patients from healthy controls.
Table IV
SELECTION OF MOLECULAR ASSAYS INCORPORATED INTO THE 51-PLEX TEST.
Assays (A) Reproducibility Direction Correlation Readoutshift <40% Distance to Assays (B) Assays (C)
of FC > 0.8 LDD >20-fold
Assays (A) selection of 22 assays was guided by (1) reproducible changes across independent cohorts of schizophrenia patients and controls (green plus; p < 0.05
in 3–5 cohorts), (2) consistent directional fold change (‘‘plus’’ sign), (3) good correlation (R > 0.8), (4) a low shift (<40%) between the first and second quality
control measurements, and (5) a high measurement to LDD ratio (>20:1) (x, lower; –, not tested). Assays (B) nine assays were selected from which the targeted
molecules are known to be involved in schizophrenia from the scientific literature or that we have identified as being differentially expressed using orthogonal
platforms. Assays (C) 20 assays were also selected for which the targeted molecules showed significant changes in bipolar disorder patients compared to controls.
288 RAUF IZMAILOV ET AL.
For the task of separating two classes (‘‘schizophrenia patient’’ from ‘‘healthy
control’’), the linear SVM (SVM with linear kernel) was selected. The linear SVM
approach considers the data as a set of 51-dimensional vectors (corresponding to
51 measured analytes) and seeks the separating hyperplane between two classes of
vectors (in our case, the class of ‘‘schizophrenia patients’’ and class of ‘‘healthy
controls’’). The hyperplane is uniquely described by a set of 51 multipliers W1,
W2, . . ., W51 and one bias term B, so that the classification of any 51-dimensional
vector {X1, X2, . . ., X51} can be done by computing the expression
S ¼ X1 W1 þ X2 W2 þ þ X51 W51 þ B
and comparing this with zero. If the result is positive, then the vector {X1, X2, . . .,
X51} is classified as an element of the first class. If the result is negative, then the
vector {X1, X2, . . ., X51} is classified as an element of the second class.
A perfect separation of two classes by a hyperplane is usually not possible,
especially if the sample size (806 subjects in this case) is larger than the dimension
of the state space (51 assays in this case). To that end, SVM allows for a certain
number of misclassification errors (a vector in the first class is classified as
belonging to the second class and vice versa) by making those errors count toward
the overall minimization goal with a penalty parameter C. Penalty parameters can
be different for both types of classification errors (one penalty C1 for classifying the
vector of the first class as belonging to the second class and another penalty C2 for
classifying the vector of the second class as belonging to the first class). Varying the
ratio of penalties C1 and C2 yields the same effect on sensitivity and specificity of
the classification SVM decision rule as moving the separation threshold in
traditional statistical decision rules. For computation purposes, we operated
here with two parameters of linear SVM: overall penalty parameter C and ratio
F of penalties C1 and C2, so that C1 ¼ C and C2 ¼ C1F.
Given a pair of parameters C and F, all elements of the data set are used for the
training of the decision rule, and performance of the decision rule was measured
using 10-fold cross-validation. For 10-fold cross-validation, the overall data set was
randomly split into 10 subsets S1, S2, . . ., S10 of equal size and then the optimiza-
tion run for each of the following 10 scenarios:
The union of S2, S3, . . ., S10 is used as a training set and the set S1 is used as a
validation set.
The union of S1, S3, . . ., S10 is used as a training set and the set S2 is used as a
validation
. set.
..
The union of S1, S2, . . ., S9 is used as a training set and the set S10 is used as a
validation set.
The measured sensitivity and specificity calculated in each of these 10 scenar-
ios was then averaged and assumed to be the sensitivity and the specificity of the
decision rule for the considered pair of parameters C and F.
ALGORITHM DEVELOPMENT FOR DIAGNOSTIC BIOMARKER ASSAYS 289
SVM-A SVM-B
Cond. probability
Cond. probability
1.0 1.0
0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.0
−10,000 −5000 0 5000 10,000 −10,000 −5000 0 5000 10,000
SVM-A score SVM-B score
100 100
80 80
60 60
%
40 40
20 20
0 0
% Accurate schizophrenia
% Accurate control
60 60
40 40
%
20 20
0 0
I II III II* I* I II III II* I*
Conditional probability region Conditional probability region
% total schizophrenia
% total control
FIG. 1. Conditional probability curves for SVM-A and SVM-B decision rules.
ALGORITHM DEVELOPMENT FOR DIAGNOSTIC BIOMARKER ASSAYS 291
for both sensitivity and specificity. The balancing was achieved by tilting the
relative weights of the corresponding false-positive and false-negative errors (the
relative weights difference is about 15% for both decision rules SVM-A and
SVM-B). The conditional probability C of class ‘‘schizophrenia’’ y ¼ 1
corresponding to the score S ¼ 0 can be made equal to 50%, but for that, the
decision rule has to be designed for maximizing the accuracy (overall error rate),
which will have unbalanced levels of sensitivity and specificity.
We use conditional probability curve to partition the decision rule output
results into five probability regions: ‘‘highly positive’’ Region I (corresponding to
conditional probabilities 100–91%), ‘‘positive’’ Region II (corresponding to con-
ditional probabilities 91–68%), ‘‘indeterminate’’ Region III (corresponding to
conditional probabilities 68–40%), ‘‘negative’’ Region IV (corresponding to con-
ditional probabilities 40–17%), and ‘‘highly negative’’ Region V (corresponding
to conditional probabilities 17–0%).
To obtain an unbiased estimate of classification performance and a biological
validation of the schizophrenia analyte signature, we determined the performance
of the decision rules by testing samples which had not been used for marker
selection in phase 1. Application of SVM-B (n ¼ 480 subjects) yielded an overall
classification accuracy of 84% (conditional probabilities are shown in Table V).
We also determined the classification accuracy of the SVM-B decision rule for
four main (positive and negative) regions of conditional probabilities (Table VI).
This resulted in an increase in accuracy of up to 96% for schizophrenia patients
and up to 97% for controls in the highest probability regions (Table VI). When
Table V
CLASSIFICATION PERFORMANCE OF SVM-B (51-PLEX DEVELOPMENT).
Accuracy estimates are shown for the entire set of samples from unique patients as well as for the subset
of 480 samples which were not used during phase 1 of the study. The conditional probability estimate is
the median of all conditional probabilities in the respective group. FE, first episode.
Table VI
CLASSIFICATION PERFORMANCE OF SVM-B (51-PLEX DEVELOPMENT).
Accuracy estimates are shown for the entire set of samples from unique patients as well as for the subset of 480 samples which were not used during phase 1 of the
study. Individual estimates are given for four main (positive and negative) regions of conditional probabilities.
The conditional probabilities in regions marked with an asterisk reflect those determined for controls.
a
Classification accuracies reflect the percentage of correct patient identifications in regions I and II and correct control identifications in regions IV and V.
ALGORITHM DEVELOPMENT FOR DIAGNOSTIC BIOMARKER ASSAYS 293
Region III was designated as indeterminate, 17% of the total number of subjects
were excluded for SVM-A, and 20% were excluded for SVM-B.
Out of the 478 total schizophrenia patients used in phase 2, 111 were suffering
from a nonparanoid type of the disease (Table VII). SVM-A identified 95 (86%) of
these patients correctly suggesting that the biomarker signature was present
regardless of the schizophrenia subtype.
We also investigated 80 subjects with a baseline Positive and Negative
Syndrome Scale (PANSS) of 66.0 17.8 before and after 4–6 weeks of antipsy-
chotic treatment. For these subjects, the treatment resulted in an overall reduction
in symptoms by 13% (average reduction of 10.3 17.3), as measured using the
PANSS positive (18% lower (average reduction of 3.7 4.1)), PANSS negative
(8% lower (average reduction of 1.6 4.9)), and PANSS general (12% lower
(average reduction of 5.0 8.5)) (Kay et al., 1987). Interestingly, application of
SVM-B led to correct identification of 85% of these patients at the first time-point
and after the treatment period. There was an average correlation of 0.49 across all
51 molecular assays, supporting the stable identification capability of the decision
rule. This suggested that schizophrenia patients in remission still feature schizo-
phrenia-like serum profiles even after 4–6 weeks of treatment.
Table VII
SUBTYPES OF THE 478 SCHIZOPHRENIA PATIENTS INVESTIGATED IN PHASE 2 OF THIS STUDY.
1. Betacellulin
2. Cancer Antigen 125
3. Calbindin
4. CTGF
5. Endothelin-1
6. IL-10
7. IL-11
8. IL-17
9. IL-7
10. MIP-1alpha
11. Thrombopoietin
Since the measurement stability of these 11 assays of the original 51-plex test
was detrimental to the overall performance of the original decision rule, these
were removed from the computation of the decision rule. In other words, while
still remaining as components of the 51-plex test for measurement and calibration
purposes, the coefficients of these assays were set to zero. As a result, it was
necessary to design a revised decision rule, which would rely only on the 40
remaining assays from the original 51-plex test.
In order to design the revised decision rule, the same combined data set drawn
from Universities of Cologne, Münster, and Magdeburg was tested with the
second lot of reagents. Due to insufficient volume of some of the samples, the
overall number of available samples decreased from 806 to 782.
The same type of decision rule optimization (search of optimal performance
among 20,100 pairs of parameters (C, F) in a rectangular grid) as described in the
previous section was carried out for this data set using the second lot of reagents.
The performance of the diagnostic decision rules was tested using 10-fold cross-
validation on the combined data set and resulted in a sensitivity of 81% and a
specificity of 81%, with ROC-AUC equal to 0.88. The conditional probability
curve and ROC-AUC for the refined decision rule are shown in Fig. 2.
1
0.9 1
0.8
0.8
0.7
Probability
0.6
Sensitivity
0.6
0.5
0.4 0.4
0.3
0.2 0.2
0.1 1-specificity
Decision rule score
0 0
−24,000 −14,000 −4000 6000 16,000 0 0.5 1
FIG. 2. Conditional probability curve and ROC for revised decision rule.
ALGORITHM DEVELOPMENT FOR DIAGNOSTIC BIOMARKER ASSAYS 295
Note that the conditional probability curve does not necessarily decrease with
the score S. For practical purposes, that should not be a problem, since the region
of nonmonotonous behavior covers the diagnostic probabilities above 90% level.
To summarize, the performance metrics of the revised decision rule closely
matched those of the original decision rule, while relying on a smaller, more
rugged subset of molecular assays from the original 51-plex test.
As described in the previous section, the refined decision rule was designed on
the second lot of reagents which caused the change of both the weight parameters
and the bias term (cutoff term) that were calculated for the original decision rule.
In order to avoid such sweeping changes of parameters for subsequent lots of
reagents, it was decided to implement a bias adjustment procedure that only
recalibrated the cutoff parameter for new lots, while retaining the values of the
weight parameters the same as they were for the refined decision rule. This bias
adjustment procedure is described in this section.
The key part of the bias adjustment procedure, which is executed at the time
of migration from an ‘‘old’’ lot of reagents to a ‘‘new’’ one, is the ‘‘calibration
pool’’—a set of samples that are measured in both ‘‘old’’ and ‘‘new’’ lots. As
described in the previous section, some of the samples had to be removed during
the transition from the first lot to the second one due to insufficient volumes. As
the migration process from one lot of reagents to another continues, the sample
volumes will decrease, and thus, for maintaining a statistically meaningful cali-
bration pool, new samples have to be added along with this migration process.
Using the calibration tool, we calculated the average measurement shift (from
the ‘‘old’’ lot of reagents to the ‘‘new’’ one) for each of the 40 assays used in the
refined decision rule. As a result, we obtained, for each analyte i ¼ 1,2, . . ., 40, its
average shift Ai, so that any calibration pool sample that is measured Xi using the
‘‘old’’ lot of reagents is expected to be measured as approximately Xi þ Ai using
the ‘‘new’’ one.
Since the decision rule score for the ‘‘old’’ lot of reagents was computed using
the formula
S ¼ X1 W1 þ X2 W2 þ þ X40 W40 þ B;
this formula, when applied to the measurements on the ‘‘new’’ lot, will give the
value
S ¼ ðX1 þ A1 ÞW1 þ þ ðX40 þ A40 ÞW40 þ B ¼ X1 W1 þ þ X40 W40 þ B ;
296 RAUF IZMAILOV ET AL.
where
B ¼ B þ A1 W1 þ þ A40 W40 :
Therefore, in order to obtain the same decision rule score on the ‘‘new’’ lot of
reagents as on the ‘‘old’’ one, we use the same weight parameters W1, . . ., W40 of
the decision rule as for the ‘‘old’’ lot, with replacement of only the cutoff
parameter B with its recalibrated value B*.
Note that the validity of this recalibration procedure is enabled by the linear
structure of the decision rule. If we had selected a nonlinear SVM, our only
recourse for changes in lots of reagents would have been the complete reoptimiza-
tion of the decision rule using the recalibration pool.
IV. Conclusions
Acknowledgments
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CHALLENGES OF INTRODUCING NEW BIOMARKER PRODUCTS
FOR NEUROPSYCHIATRIC DISORDERS INTO THE MARKET
Abstract
I.Introduction
II.Biomarker Blood Tests for Diagnosis and Management of Mental Disorders
III.Current Dilemmas in Psychiatric Diagnosis
IV. The Potential for Biomarker-Based Diagnostic Tests in Psychiatry
V. Why Is Early Diagnosis So Important?
VI. Historical Perspective—The Blood of the ‘‘Insane’’
VII. Biomarkers: Not Quite Living up to the Promise?
VIII.Biomarkers: What Are the Issues?
A. Regulatory
B. Technologies
C. Strategies
D. The Problem of Size
E. The Problem of Acceptance
IX. Development of a Molecular Blood Test for Schizophrenia
X. Conclusions
Acknowledgments
References
Abstract
There are many challenges associated with the discovery and development of
serum-based biomarkers for psychiatric disorders such as schizophrenia. Here, we
review these challenges from the point of view of psychiatrists, general practitioners,
the regulatory agencies, and biomarker scientists. There is a general opinion in
psychiatric medicine that improvements over the current subjective tests are essen-
tial. Despite this, there is a reluctance to accept that peripheral molecules can do the
job any better. In addition, psychiatrists find it difficult to accept that peripheral
molecules, such as those found in blood, can reflect what is happening in the brain.
However, the regulatory health authorities now consider biomarkers as important
for the future of drug development and have called for efforts to modernize methods,
tools, and techniques for the purpose of developing more efficient and safer drugs.
We also describe here the development of the first ever molecular blood test for
schizophrenia, and its reception in the market place, as a case in point.
I. Introduction
Biomarkers for risk Biomarkers for early Biomarkers for prognosis, side
prediction detection effects, and drug response
This requires that molecules must achieve the status of validated biomarkers to be
used in regulatory decisions for clinical trials. The FDA has now established three
types of biomarkers: (1) exploratory biomarkers, (2) probable valid biomarkers,
and (3) known valid biomarkers (Goodsaid and Frueh, 2007). For the first class,
there must be scientific evidence for proof of concept. The second class requires
that biomarkers can be measured in an analytical test system with strict perfor-
mance characteristics and that there is established scientific evidence that explains
the significance of the results. The third class requires replication of the results at
different sites, for cross-validation purposes.
However, this is not a simple task. The identification of biomarkers for
psychiatric disorder diagnostics is challenging due to the poor understanding
and delineation of these conditions using the current subjective methods, the
overlap of symptoms across different disorders, and marked heterogeneity across
human subjects. However, emerging proteomic platforms have facilitated the
identification of biomarkers by simultaneous measurement of hundreds or
thousands of molecules in nonhypothesis-driven profiling studies. Our group
has recently employed an approach based on multiplexed immunoassay profiling
which resulted in identification of a serum signature that could identify schizo-
phrenia patients with an accuracy of 82% across five independent patient cohorts
(Schwarz et al., 2010).
In this chapter, we discuss the challenge of bringing a molecular test for
neuropsychiatric disorders to the market. The first part discusses the general
problem of introducing the new paradigm of molecular biomarkers into the
conventionally nonmolecular field of psychiatry. The second part discusses the
associated challenges and methods for the identification of biomarkers. In partic-
ular, we elaborate on the potential uses of molecular biomarkers in the field of
psychiatric disorders, particularly for improved clinical classification and manage-
ment of patients and as a means of facilitating the process within the pharmaceu-
tical industry for improved drug discovery.
II. Biomarker Blood Tests for Diagnosis and Management of Mental Disorders
Table I
LIST OF CLINICAL UTILITIES FOR DISEASE BIOMARKERS OF PSYCHIATRIC DISORDERS.
Table II
LIST OF PROTEIN/GENE EXPRESSION ASSAY TESTS FOR A RANGE OF INDICATIONS.
the prevalence of schizophrenia when ICD-10 criteria are used for diagnosis,
compared with the use of DSM-IV (Cheniaux et al., 2009). This may be because
DSM-IV and ICD-10 have developed to include a modern compendium of
mental disorders that can be reliably diagnosed based on signs and symptoms,
but have not been validated (Spitzer et al., 1980; Pierre, 2008; Keller et al., 2011). It
is not likely that specific symptoms are linked to a defined natural disease entity. It
is well known that patients with neurological, traumatic, infectious, and metabolic
disorders can present with symptoms indistinguishable to symptoms of schizo-
phrenia (Yolken et al., 2009; Lovatt et al., 2010; Scaglia, 2010). In addition, some
subjects are known to have feigned symptoms of schizophrenia and other mental
disorders (Bagby et al., 1997) for reasons such as gaining access to disability
payments, social housing, and other benefits.
Most psychiatrists agree that the current construct of schizophrenia is an
umbrella term for a complex chimera of etiologies that happens to present with
similar symptoms, in the same way that most acute infectious disorders present
with fever (Tsuang, 1975). Misdiagnosis is thus a common occurrence in psychi-
atric practice. For example, Gonzalez-Pinto et al. (1998) found that 31% of bipolar
patients were diagnosed with schizophrenia. Follette and Houts (1996) went
further in their criticism challenging the fundamental assumptions or theoretical
underpinnings of current classifications systems. They pointed out that there is no
method to validate current diagnostic concepts with externally validated measures
which are independent of the concept itself.
A further potential factor for misdiagnosis and inconsistency is that clinicians
do not usually use classification systems to establish a psychiatric diagnosis. In-
stead, they mostly apply heuristic unstructured interviews. This means their
diagnosis may be based on experience and personal views, instead of matching
the guidelines or criteria of the diagnostic system. This can be associated with
systematic errors in judgment based on misconception and experience, which may
rely on selective memory. There has also been a failure to address the problem of
false positives in diagnoses of mental disorders (Wakefield, 2010). A study pub-
lished by Strakowski et al. (2003) investigated the influence of ethnicity on patient
diagnosis. This study found that clinicians tend to overdiagnose schizophrenia in
African Americans. The bias was removed when examiners were provided with
ethnicity-blinded transcripts of otherwise identical patient interviews.
The concordance rate for identical twins to develop schizophrenia lies be-
tween 11% and 69%, according to different studies (Torrey, 1992; McGue, 1992;
Tsuang, 2000). Such twin studies provide indisputable evidence that a genetic
component and predisposition to develop schizophrenia exists. However, it also
means that, even when such a predisposition exists (as in identical twins), an
individual will not necessarily develop schizophrenia. Environmental and other
nongenetic factors appear to play a more important role in most patients. A range
of environmental factors could affect brain function in subjects with psychiatric
illnesses. These could include pregnancy and delivery complications, such as
intrauterine hypoxia, infections, and malnutrition (Dauncey and Bicknell, 1999;
Schlotz and Phillips, 2009). There are also nonbiological factors which could
precipitate the onset of mental illness, including psychosocial stressors such as
experiencing natural disasters, loss of a family member or close friend, residence
in a poor or dangerous area, or experiencing a dysfunctional family life (Koenig
et al., 2002). It is likely that such environmental factors can interact with genetic
components in a negative manner in the development of psychiatric conditions.
This means that disease prevention or minimization might be possible if
discrete environmental risk-factors can be determined. Above, we indicated the
occurrence of metabolic abnormalities such as insulin resistance in a proportion of
schizophrenia patients. We and other researchers have estimated this proportion
to be 20–50% of in first-onset subjects (Ryan et al., 2003; Spelman et al., 2007; van
Nimwegen et al., 2008; see Chapter ‘‘Abnormalities in metabolism and hypotha-
lamic-pituitary-adrenal axis function in schizophrenia’’ by Guest et al.). In addi-
tion, several researchers have indicated the presence of circulating inflammatory
and immune response factors in first-onset schizophrenia patients (Szulc et al.,
2001; Riedel et al., 2005; van Venrooij et al., 2010). In a preliminary study, we have
shown that various markers relating to these subgroups can be identified in
patients even prior to disease onset (Schwarz et al., in press). This study analyzed
sera obtained from U.S. military personnel approximately 30 days before the
onset of symptoms. An important hypothesis to test will be whether or not disease
CHALLENGES OF INTRODUCING NEW BIOMARKER PRODUCTS 307
Table III
ESTIMATED BENEFITS OF EARLY DIAGNOSIS AND TREATMENT OF SCHIZOPHRENIA.
The data are based on current diagnosis and compared to the established disease phase (Davies and
Drummond, 1990; Knapp et al., 2004; Wu et al., 2005; Hafner and Maurer, 2006).
308 SABINE BAHN ET AL.
only Bleuler’s term and not his original disease concept survived into our time.
For more than a century, changes in pituitary function and related hormonal
abnormalities in various psychiatric disorders have been established and
remain a strong biological finding today. Endocrinology also provided a direct
analogical bridge which led to the discovery of the first neurotransmitter
(neurohormone), acetylcholine, by German biochemist Otto Loewi in 1921.
3. The immunoserodiagnostic paradigm (1906)
The development of the Wasserman reaction test for neurosyphillis in 1906 was
arguably the first great breakthrough in biological psychiatry. It was the first
diagnostic blood test for a discrete form of insanity (general paralysis of the
insane) commonly observed in asylums. The discovery inspired other immuno-
logical investigations. In 1909, two German psychiatrists injected cobra venom
into patients with dementia praecox and manic-depressive insanity and reported
that all of the former, and only some of the latter, evidenced blood serum
reactions to the toxin, and healthy people did not. However, the so-called
Much-Holzmann psycho-reaction could not be replicated and was quickly
refuted. A much more influential immunoserodiagnostic test developed by the
prominent Swiss biochemist Emil Abderhalden (1877–1950), the ‘‘defensive
ferments reaction test,’’ was found by the German psychiatrist August Fauser
(1856–1938) to differentially diagnose dementia praecox and manic-depressive
insanity from healthy subjects in a series of studies. From December 1912 to
perhaps as late as 1920, many in the international scientific community believed
a valid blood test for madness had been found. However, beginning in 1914, a
series of studies were published that could not verify the existence of Abderhal-
den’s defensive ferments (Abwehrfermente), and not only the test but also its
usefulness to psychiatrists were cast into doubt. In the past century, changes in
immune function and inflammation have been linked to various psychiatric
disorders through many lines of evidence (see Chapter ‘‘Immune and neuro-
immune alterations in mood disorders and schizophrenia’’ by Drexhage et al.).
4. The medical genomics (2005)/postgenomics (2010) paradigm
Twenty-first-century blood tests have targeted both the genome and the prote-
ome. However, attempts to specify most medical diseases (especially neuropsy-
chiatric disorders) at the level of the genome have been disappointing. The
hereditary component of some (mostly severe) psychiatric disorders has now
been established beyond doubt. However, there is a lack of agreement as to how
strong the genetic and environmental contributions are and how they interact to
precipitate the onset of severe mental illness. For example, despite 20 years of
profound and expensive efforts, no single gene or combination of genes has
been identified that substantially increase the probability of developing schizo-
phrenia. In 2009, results from the largest Genome Wide Association Studies
310 SABINE BAHN ET AL.
The field of clinical proteomics has raised high hopes generated by reports on
potential biomarkers. In most cases, these could not be substantiated in validation
studies or in clinical trials. Potential reasons for the failure to link biomarkers into
clinical studies include deficiencies in design and analysis, the problem that drug
targets and biomarkers may not be causal to the disease but rather a result of the
disease process, a lack of congruence in the animal models of the disease with the
human condition, or the enrolment of patients in clinical trials who are too
advanced in disease stage to show any response to potential therapeutics (Flood
et al., 2011). In the case of Alzheimer’s disease, a consensus has now been reached
for testing drug candidates in the earlier stages of the disease (Aisen et al., 2011).
The suggestion that biomarker research has not lived up to the initial hype has
been demonstrated by the fact that multiple ‘‘breakthrough’’ biomarkers have
been publicized but have not reached the market place. Apart from some bio-
markers in the field of cancer research, most have not been validated and have
faded from the spotlight. Major cancer biomarkers that have received FDA
approval over the past few decades include prostate-specific antigen for prostate
cancer (Kuriyama et al., 1980), carcinoembryonic antigen (CA)-125 for ovarian
cancer (Klug et al., 1984), and CA-19-9 for pancreatic cancer (Satake et al., 1985).
However, most of these biomarkers are used mainly for monitoring treatment
response and are not suitable for early diagnosis with the exception of prostate-
specific antigen (Gjertson and Albertsen, 2011).
The Human Proteome Organization (HUPO) emerged from the Human
Genome Project as a means of understanding gene function. HUPO has devel-
oped several initiatives targeted at overcoming the problem of reproducibility, as a
major problem has been the fact that most proteomic researchers have been
unable to reproduce their data. These initiatives are focused on plasma, liver,
brain, disease glycomics/proteomics, disease biomarkers, mouse disease models,
model organisms, kidney/urine, cardiovascular disease, stem cells, the Human
Antibody Initiative, and the Proteomics Standards Initiative (http://hupo.org/).
Each of these initiatives is based in one country and includes subprojects involving
CHALLENGES OF INTRODUCING NEW BIOMARKER PRODUCTS 311
international partner laboratories. The rationale for this structure is that most of
the reproducibility problem is likely to be due to several sources of variability
including the biological sample, sample handling, study design, technical and
user-related differences. However, the first proteomics-based test approved by the
FDA, the OVA1 ovarian cancer triage test (Quest Diagnostics), demonstrates that
this is possible (Zhang and Chan, 2010).
A. REGULATORY
Regulatory
approval
signal and the clinical result will lead to a more efficient and less risky develop-
ment process. Therefore, inclusion of biomarkers into clinical development will
only be achieved by a rigorous scientific approach, including standardized sample
collection, analysis, and data processing. Early interaction with the appropriate
regulatory agencies is advised to ensure that studies are designed and biomarker
tests are carried out appropriately. One of the best examples of a codeveloped
biomarker and drug combination is that of the HER2/neu subtype of epidermal
growth factor receptor (EGFR) with the humanized monoclonal antibody trastu-
zumab (HerceptinTM; Taube et al., 2009), as mentioned above. In this case, breast
cancer subjects who overexpress the HER2 subtype of EGFR are more likely to
respond to HerceptinTM treatment (Pegram et al., 1998). The example of HER2/
HerceptinTM and other biomarker/drug combinations involving the use of scien-
tifically and analytically validated biomarkers and rationally designed hypothesis-
testing may lead to a paradigm shift in clinical trials, as appears to be the case in
the field of cancer therapeutics (Tan et al., 2009).
The European health authorities have also initiated activities to support
the development and implementation of biomarkers through agencies such as the
Innovative Medicines Initiative (Kamel et al., 2008; Hunter, 2008). The Innovative
Medicines Initiative is a partnership between the European Commission and the
pharmaceutical industry that aims to promote more efficient discovery and develop-
ment of medicines by supporting research into the drug development process. One
of the main objectives is the discovery of translational biomarkers, including for
psychiatric conditions such as schizophrenia and autism spectrum disorders. The
European Commission contributes one billion euros to the program and this
amount is matched by in kind contributions consisting mostly of research activities
worth an equivalent amount from member companies of the European Federation
of Pharmaceutical Industries and Associations (EFPIA).
B. TECHNOLOGIES
In the case of genetic linkage studies for psychiatric disorders, there have been
many linked loci which were claimed and withdrawn, many association studies
published and not confirmed, and many new and different chromosomal regions
implicated for the same disorders. Thus, a lack of reproducibility in other linkage
and association studies has generated considerable doubt from scientists that only
a concerted effort will be able to rectify. Without this, it is unlikely that such
knowledge will be translated into the clinic (Bondy, 2011). These results have led
to skepticism from clinicians, scientists, and regulatory agencies, which will make
the introduction of valid biomarkers into clinical diagnostics or the drug discovery
industry even more difficult (Kopec et al., 2005).
314 SABINE BAHN ET AL.
(Duan et al., 2008), coronary arterial disease (Gurbel et al., 2008), inflammatory
response after cardiopulmonary bypass (Aĝirbaşli et al., 2010), schizophrenia
(Schwarz et al., 2010), and a phase II study of urothelial cancer therapy
(Bellmunt et al., 2011).
C. STRATEGIES
It is now generally accepted that single biomarkers are not likely to be effective
given the fact that the complexity of most diseases, particularly psychiatric
disorders, cannot be represented by a single marker (Boja et al., 2011). Also,
most psychiatric conditions appear to be the result of a complex interaction
between environmental and genetic factors (Dick, 2011). Therefore, a panel of
biomarkers must be employed to reflect this complexity and to add specificity to
the measurements. However, it is now becoming clear that such biomarker panels
must consist of rigorously validated molecules in multiple centers and across
different time points in order to provide a reproducible and accurate test.
However, in most cases, biomarker candidates have failed at this hurdle
(Alymani et al., 2010).
Biomarker panels must also be disease specific, at least relative to other
diseases which have similar symptoms. Again, this is particularly difficult for
psychiatric disorders as these have many areas of overlap of subjectively assessed
behavioral symptoms. Examples for this are the overlap of negative symptoms
between schizophrenia and major depressive disorder (Fleischhacker, 2000), the
similarity in psychotic symptoms between manic bipolar disorder and schizophre-
nia (Dunayevich and Keck, 2000), and the shared cognitive deficits across all of
these conditions (Ferrier et al., 1999). In addition, conditions such as bipolar
disorder are characterized by having multiple stages such as the cycling of
manic and depressed moods (Dunayevich and Keck, 2000). Identification of
valid biomarkers which could predict the switch from one stage to another
would be invaluable.
Psychiatric conditions also come with the special challenge that they have
traditionally been considered to be disorders of the mind. Thus, convincing clin-
icians and the regulatory agencies that blood-based assays are sensible and can be
predictive is most likely the biggest challenge of all. However, the evidence is
accumulating that such conditions are essentially systemic disorders and not restrict-
ed to the brain. For example, several hormones secreted from the diffuse neuroen-
docrine system are now known to be altered even in first-onset schizophrenia
patients. These include hormones from pancreatic islets, the pituitary, gonads,
adrenal glands, the gastrointestinal system, and adipose tissue (Yang et al., 2008;
Schanze et al., 2008; Venkatasubramanian et al., 2010; Guest et al., 2010, 2011).
316 SABINE BAHN ET AL.
Post
Protein Protein Biological
DNA RNA Protein translational
complex network effect
modification
FIG. 3. The complexity of the human proteome in the production of biological effects.
318 SABINE BAHN ET AL.
the field of psychiatric disorders than in other medical disciplines. This is despite
the fact that schizophrenia patients are more likely to develop other conditions
such type II diabetes mellitus (15.8%) compared to the general population
(2–3%), and this has been known for the past 40 years (Dynes, 1969; Tsuang
et al., 1983). Alterations in the prevalence of various infectious and immunological
disorders, such as celiac disease have also been reported for approximately the
same duration (Dohan, 1970).
At the time of the launch of VeriPsychTM in the United States, leading
psychiatrists were asked whether they believe a blood test can really detect a
mental illness. The following general attitudes and answers were provided:
(http://www.msnbc.msn.com/id/39686973/ns/health-mental_health/). Some
physicians were skeptical of the test. While recognizing the need and potential
for relying on methods other than patient reports or observations, ‘‘the science is
not there yet,’’ said Dr. Gregory Light, an associate professor of psychiatry at the
University of California, San Diego. ‘‘Blood-based tests, at the moment, are far
away from being useful for an individual patient,’’ Light said. ‘‘For example,
genetic tests raise more questions than they answer, account for only a small
number of cases, and the advances in technology are rapidly outpacing our ability
to interpret the results.’’ There appears to have been a misunderstanding that the test under
discussion is based on genes, when it is actually based on proteins and small molecules as a
multiplexed biomarker blood test. ‘‘The biomarkers that some research has suggested
are associated with schizophrenia might be a consequence of the illness, or long-
term use of antipsychotic medication,’’ said Dr. Irving Gottesman, a professor
emeritus of psychology at the University of Minnesota. As mentioned above, biomarkers
that are a response to underlying illness can lead to development of valuable biomarker tests, such
as prostate-specific antigen for prostate cancer and troponin for cardiac injury. "It takes, often, a
period of observation, both with and without medication of 6 months or more, to
get a better ‘‘feel’’ for what may be the proper diagnosis," Gottesman said.
However, by the time the psychiatrist has a better feel for the disorder, the patient’s life and life
quality may have been irreversibly devastated, as described above.
0.6 2
Severity of symptoms
tom itive
1
1 Poor outcome
mp os
s
0.5
sy st p
Fir
0.4
0.3
Better outcome
0.2
Prodrome phase
0.1
0
Time from prodrome phase to diagnosis > 3–8 years
X. Conclusions
This chapter has described the many challenges associated with the discovery
and development of blood-based biomarkers for psychiatric disorders. The current
diagnostic process and strategies for developing novel pharmaceutical compounds
are in need of an overhaul. As stated by the Nobel Laureate Lee Hartwell at the
2004 HUPO meeting in Beijing, ‘‘Biomarkers for early diagnosis will revolutionize
the pharmaceutical industry allowing diseases to be treated at an earlier stage—
increasing survival rate.’’ Despite this, there is a reluctance to accept the idea that
biomarkers will be of any help at all (Poste, 2011). It is true that only a handful of the
thousands of promising biomarkers identified have lived up to the initial hype.
However, the regulatory health authorities now consider biomarkers as important
for the future of drug development and have called for efforts to modernize
methods, tools, and techniques to achieve this. As U.S. President John Fitzgerald
Kennedy said in 1962, ‘‘we will go to the moon because it is hard.’’ It is interesting
that in the early 1960s, many of the technologies required had not even been
invented. Not only that, a massive integration of technologies was required to
produce what many have claimed is the finest achievement of the twentieth century.
There is now reason for optimism that further technological advancements, and
interdisciplinary approaches will overcome current limitations in the field biomar-
kers to help usher medicine fully into the twenty-first century.
Acknowledgments
This research was supported by the Stanley Medical Research Institute (SMRI),
the European Union FP7 SchizDX research programme (grant reference 223427),
and the NEWMEDS Innovative Medicines Initiative. We also thank Enrique
CHALLENGES OF INTRODUCING NEW BIOMARKER PRODUCTS 321
Millan for research gathered through his MBA individual project entitled The Value
of the Schizophrenia Diagnostic Market, carried out with the Judge Business School at
Cambridge University and Psynova Neurotech in Cambridge, UK.
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TOWARD PERSONALIZED MEDICINE IN THE
NEUROPSYCHIATRIC FIELD
Erik H.F. Wong1, Jayne C. Fox2, Mandy Y.M. Ng2 and Chi-Ming Lee3
1
AstraZeneca Pharmaceuticals, External Science, CNS-Pain Innovative Medicine Unit,
Wilmington, Delaware, USA
2
AstraZeneca Pharmaceuticals, Personalized Health Care and Biomarkers, Alderley Park,
Macclesfield, Cheshire, United Kingdom
3
AstraZeneca Pharmaceuticals, Discovery Enabling Capabilities & Sciences, Alderley Park,
Macclesfield, Cheshire, United Kingdom
Abstract
I. Introduction
II. Is Personalized Medicine All About Genetics? How Many Measures Are We Talking About?
A. Heritability, Structural Genomics, and Personalized Medicine
B. Genetic Approaches for Population Stratification to Avoid False Associations
C. Gene and Environmental Approach for Personalized Medicine
D. Advanced Technical Development in Genomics
E. From Biology Advance to Technology Challenge
III. Opportunities for Personalized Medicine in Neuropsychiatry for Drug Discovery
A. Biomarker Technology Beyond Genetics
B. The Challenges of PM in Neuropsychiatry—A Lesson from Other Fields
C. Endophenotypes—Leveraging Biology for Psychiatric Drug Discovery and Development
D. Pharmacokinetics and Personalized Medicine
E. Biomarker Discovery/Qualification/Validation—The Partnership Model
IV. Conclusion
Acknowledgments
References
Abstract
regulatory approvals have precipitated a drug pipeline crisis and eroded confi-
dence in the industry. This chapter describes how innovations in genomics and
translational medicine can impact the future of neuropsychiatry and deconvolute
the complexity of psychiatric diseases from symptoms biology. A targeted and
consistent investment is needed to restore confidence in translating science into
clinical success.
I. Introduction
II. Is Personalized Medicine All About Genetics? How Many Measures Are We Talking About?
Disease states are multifactorial in etiology and cannot be assessed solely using
genetics. Uher (2009) reported that the heritability of different mental illnesses is
variable, from 0.3 (anxiety disorders) to 0.9 (autism; see Table. I). However, the
understanding of the interplay between genetic and nongenetic factors is still
unclear for most mental illnesses, regardless of disease prevalence.
The assumption that heritability of a disease state will necessarily open doors
for direct, genetics-based, drug treatment decisions is reductive because the space
between clinical phenotype and genotype is exceedingly complex. However, the
potential for genetics-based approaches to understand disease processes will
provide important insights is indisputable (Green and Guyer, 2011). Such discov-
ery will provide a route for the personalized approach of drug treatments. Genetic
approaches may not provide direct routes for personalization, that is, via a ‘‘genetic
test,’’ as was originally proposed, but rather—particularly in the neuropsychiatric
field—genetic approaches will help to unravel the complexity of the underlying
diseases, thereby providing a scaffold for future biomarker approaches. This is
exemplified by the evolving field of structural genomic variation. Structural varia-
tion was considered to be less important in the early days of the HGP than the
potential role played by common, simple variations (single nucleotide polymorph-
isms, SNPs) either as direct markers or ‘‘tags’’ for coinherited sequence blocks
(International HapMap Consortium, 2005). Technological advances have allowed
genomic studies to move beyond common, simple genetic variations to identify
TOWARD PERSONALIZED MEDICINE IN THE NEUROPSYCHIATRIC FIELD 333
Table I
GENETIC EPIDEMIOLOGY OF SELECTED TYPES OF MENTAL ILLNESS.
Lifetime prevalence (percentage), median age of onset (years), mortality ratio (values of more than one
indicate increased mortality compared with the general population), fertility ratios (values less than one
indicate decreased fertility compared with the general population), heritability (estimated contribution
of addictive genetic effects from twin studies), and an index of paternal age effect (risk ratio for 10-year
increase in fathers aged above 30 years; no data are available for anorexia nervosa and anxiety
disorders). All data are based on published report referenced in the ‘‘Building Blocks’’ section of
Uher (2009).
therapies. This has been recently reviewed and discussed (Klein, 2011; Grinker,
2010). Undoubtedly, if empirical analyses of DSM data give rise to meaningful
treatment subgroups, then such opportunities should certainly be grasped. How-
ever, we believe that additional molecular or imaging-based biomarkers will be
needed and large international consortia are now attempting to identify such
markers. This is illustrated by the Sequenced Treatment Alternatives to Relieve
Depression (STAR*D; Rush et al., 2004) study funded by the National Institute of
Mental Health (NIMH) in the United States, and the Innovative Medicine
Initiative (IMI)-Novel Methods Leading to New Medications in Depression and
Schizophrenia (NEWMEDS) study funded by the European Commission
(Hughes, 2009; Abbott, 2010). By adopting an open approach to the exact nature
of potential stratification tools—using the best platforms and systems available,
but not focusing exclusively on a given technical approach—it seems feasible that
markers deriving from different discovery modes could be combined and ulti-
mately assayed via a single diagnostic platform, much as many common biochem-
ical markers are measured in routine clinical decision making (Morrow et al.,
2007). Obviously, this approach would be a complex solution, and it would be
preferable to devise simpler ways to personalize or segment neuropsychiatric
disease populations and thus derive improved treatment outcomes. Attempts
have been made in this area, in particular, to subtype depression (Parker et al.,
1999), although the application of an agreed common approach to personalize
treatment choices, beyond current clinical practice, is not apparent.
The solution to one problem often unveils another. By applying the squeezed
pipeline/moving bottle neck analogy, it is clear that the bottle neck has moved
from sequence generation to data storage, computational power, and statistical
analysis. Next-generation sequencing or contemporary SNP arrays are an in-
tensely data-driven technology. For example, the Illumina Human Omni 2.5 can
assay 2.5 million variants with minor allele frequencies down to 2.5% and the
1000 genome project has identified 20 million unique variants in 629 individuals
(http://www.1000genomes.org/). The integration of this immense volume of data
with clinical phenotypes presents significant challenges in statistics, data storage,
and computational power. SNPs are often analyzed one at a time in the current
GWAS studies. However, many researchers have advocated a more holistic
approach that incorporates gene–gene and gene–environment interactions. The
billions of comparisons of SNP pairs present a significant challenge in computa-
tion and interpretation of results due to problems of multiple testing. Substantial
investments are required to build and maintain a computational infrastructure,
including computing cluster and the relevant utilities for cluster operation.
336 ERIK H.F. WONG ET AL.
findings that relate to enrichment for structural genomic variants in both schizo-
phrenia and other neuropsychiatric disorders (Grozeva et al., 2010). Although
these new findings suggest some overlap in the genetic basis of neuropsychiatric
conditions, the frequency of the individual genetic changes in disease populations,
together with the impact of other genetic and nongenetic factors (Girirajan and
Eichler, 2010), likely indicates their best use as tools to unravel biological pathways
rather than stand-alone biomarkers for treatment decisions.
There are some success stories for personalized medicine in oncology includ-
ing Herceptin in HER2þ breast cancer, Gleevec in chronic myeloid leukemia,
CML, and Rituxan in chronic lymphocytic leukemia, CLL. Herceptin was
approved for breast cancer patients with an overexpression of HER2, human
epidermal growth factor receptor ErbB2 (Ross et al., 2009), and response to
Gleevec is indicated for CML patients expressing BCR-ABL, the fusion protein
created by a translocation between the long arms of chromosomes 9 and 22
(Druker, 2008). However, this technological progress in solid and liquid tumors
has not been mirrored in personalized medicine in neuropsychiatry.
The success of personalized medicine in oncology is dependent on matching
drugs with patients whose cancer is caused by the mechanism targeted by the
drug. However, in psychiatry, the etiology and pathophysiology of many disorders
are largely unknown and the categorization of psychiatric diseases using DSM/
ICD criteria is largely based on assessment of symptoms and generally lacks
proven biological validity (Klein, 2011). Most pharmacotherapy in psychiatry is
discovered empirically based on serendipitous clinical observation and reversed
pharmacology (Wong et al., 2010b). Thus, there is insufficient understanding of
disease biology to inform diagnosis and guide treatment selection for most
neuropsychiatric disorders. Further, traditional pharmacotherapy in psychiatry
treats patients with a ‘‘one-size fits all’’ broad spectrum population approach. It is
perhaps not surprising that there is a low-treatment response rate; for any given
psychiatric drug on the market works on average for only about half of the patients
who receive it. For instance, the remission rate for MDD and bipolar disorder
after conventional pharmacotherapy is only about 30% (Warden et al., 2007;
Thase, 2008).
It is also increasingly recognized that not all psychiatric patients with the same
DSM/ICD diagnosis respond equally to the same treatment. The difference in
response of different patients to a particular drug may be due to different genetic
factors and/or patients’ age, diet, or other environmental issues (such as psycho-
logical stress), and models are being used to tease apart these potential contribut-
ing components (Oliver and Davies, 2009). It is not unusual to have a
338 ERIK H.F. WONG ET AL.
treatment approach and existing clinical trial designs—the need for personalized
medicine is obvious and urgent. This latter point is an important driver for the
real cross-pharma (often referred to as ‘‘precompetitive’’) and globally coordinat-
ed efforts for better, therapy-linked patient segmentation, as exemplified by EU
Innovative Medicine, Novel Methods leading to New Medications in Depression
and Schizophrenia, that is, IMI-NEWMEDS (Abbott, 2010). In addition, efforts
by individual companies suggest an opportunity for fresh thinking is now being
embraced.
IV. Conclusion
It is clear that the ‘‘moment in the sun’’ for personalized medicine remains
elusive for neuropsychiatry. The complex genetic and nongenetic factors that
influence psychiatric disorders point to a need for a number of technologic
breakthroughs in biology and organizational approaches. It has taken a crisis of
confidence in neuropsychiatric research for psychiatric drug discovery and devel-
opment to drive the realization that these challenges cannot be overcome at the
individual lab or company level. The message here is clear; the scale of the
challenge, and consequent investment requirement, has driven the need for
precompetitive consortia that leverage open innovation to address the issue
mentioned above. We remain optimistic that the formation of IMI, and similar,
efforts signal a turnaround in approach to enable this form of ‘‘BIG’’ science and
that step by step, the rules of engagement that connect neuropsychiatric pheno-
type to molecular etiology will be revealed to allow our research community to
innovate back to the future!
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CLINICAL UTILITY OF SERUM BIOMARKERS FOR MAJOR
PSYCHIATRIC DISORDERS
Abstract
I. Introduction and Scope of the Subject
A. What is a Biomarker?
B. Focus on Serum Markers for Major Psychiatric Disorders
C. Clinical Examples
D. The Potential Use of Biomarkers in These Cases
II. About the Concept of Diagnosis in (Current) Psychiatry
III. What Should/Might a Useful Biomarker Predict/Distinguish in Psychiatry
A. A Diagnostic Category
B. A Subgroup Within a Diagnostic Category
C. A Specific Treatment Response
D. Novel Diagnostic Categories Overlapping the Boundaries of Traditional
Diagnostic Categories
E. Specific Developmental Trajectories (i.e., Clinical Staging Based on Biomarker Profiles)
IV. Intermezzo: Lessons from Oncology
V. Overview of Current Results on Serum Biomarkers
A. Single Molecular Measurements
B. Multiplex Molecular Measurements
VI. Future Prospects, Research Agenda
Acknowledgments
References
Abstract
for psychosis and existing patients who are likely to progress to more severe states.
The authors argued for the use of broader categories of related patients and to
deconstruct the traditional diagnoses in favor of molecular biomarker profiles.
A. WHAT IS A BIOMARKER?
In psychiatry, the term biomarker has been used for virtually every nonsubjective
measurement, ranging from imaging data [functional or structural magnetic reso-
nance imaging (MRI)], neuropsychological tests (i.e., the test for sustained attention),
electrophysiological responses (i.e., the P300 event related potential wave), or even
skin flush after chemical provocation (Horrobin, 1980; Smesny et al., 2007). In this
chapter, we focus on the use of molecules identified in peripheral blood (i.e., proteins
or gene-expression data). Further, we focus on the major psychiatric disorders:
schizophrenia, bipolar disorder, and major depressive disorder.
CLINICAL UTILITY OF SERUM BIOMARKERS FOR MAJOR PSYCHIATRIC DISORDERS 353
C. CLINICAL EXAMPLES
mankind is threatened’’ and that ‘‘everybody should listen to her warnings and
improve their lives.’’ She also said that she received messages from the president of
the United States through the television and heard his and other voices giving her
advice about the future of the world. She was diagnosed with schizophrenia and
treated with the antipsychotic quetiapine. This resulted in disappearance of the
delusions and hallucinations, but the patient became slower and psychomotori-
cally retarded, and talked less, apart from that ‘‘there is no future anymore.’’
A second diagnosis was then made: ‘‘negative symptoms of schizophrenia, possibly
depression.’’ Consequently, an antidepressant was added to the medication. After
1 week, the patient became agitated and restarted her statements about being a
prophet. She also showed sexual disinhibition and picked up men in bars. After
admission in a psychiatric hospital, she wore excessive makeup and could not stop
talking. The diagnosis was then changed to a manic episode in the course of either a
bipolar or a schizoaffective disorder. The antidepressant was stopped and a mood
stabilizer was given. After 3 weeks, there was almost complete recovery.
3. Another Young Female with Schizophrenia
A 19-year-old college student with no psychiatric history and in general good
health developed auditory and visual hallucinations over the course of 2 weeks.
She was referred to a general mental health-care facility by her general practi-
tioner. A provisional diagnosis of ‘‘psychotic disorder not-otherwise-specified
(NOS),’’ possibly with concomitant ‘‘personality disorder,’’ was made and treat-
ment with olanzapine was started. After 1 week, hallucinations were still present
and delusions developed; the patient thought that her parents were poisoning her
food. A couple of days later, the patient developed acute anxiety and severe
agitation, prompting an acute forced admission into the psychiatric hospital.
At admission, several clinical diagnoses were considered: ‘‘psychotic disorder
NOS,’’ schizophreniform disorder/early phase schizophrenia, major depressive
disorder, and psychotic/mood phenomena accompanying a personality disorder.
Routine neurological and laboratory examination was performed, but no aberra-
tions were apparent. Over the next weeks, a gradual amelioration occurred,
although the patient showed impaired concentration and (according to her
parents) ‘‘altered personality’’ and ‘‘odd mood swings.’’ A psychological test
after partial remission showed deficits in working memory and sustained atten-
tion. A final diagnosis of ‘‘paranoid schizophrenia’’ was made. Seven months after
the first symptoms, the patient again developed anxiety, and odd movement
disorders described as ‘‘catatonia.’’ She was readmitted into the psychiatric
hospital with a ‘‘second psychotic episode.’’ After a few days, she displayed
‘‘disorientation’’ and ‘‘disorganized speech.’’ An electroencephalogram testing
showed signs of ‘‘encephalopathy’’ but no clear epileptic activity. Brain MRI
results appeared normal. The diagnosis was reformulated in ‘‘psychosis/schizo-
phrenia’’ with status epilepticus, possibly a ‘‘somatic syndrome.’’
CLINICAL UTILITY OF SERUM BIOMARKERS FOR MAJOR PSYCHIATRIC DISORDERS 355
To fully grasp the possibilities as well as the issues related to the possible use of
biomarkers in psychiatric practice, it is important to have an understanding of the
concept of a diagnosis in medicine and, in particular, psychiatry. It will also be
important to have an understanding of recent developments related to diagnostic
concepts in psychiatry.
Any form of rational medicine depends on the existence of a valid method to
group similar patients under a diagnosis. At present, the major psychiatric
diagnostic system is the widely used Diagnostic and Statistical Manuel (DSM)
system (Sadler, 2005), with the 1994 DSM-IV as the most recent edition.
The revised DSM-V is expected in 2012.
Although the influential DSM-III was introduced in 1980, its fundamental
structure tracks back to the late nineteenth and early twentieth centuries, when
Kraepelin (1971) made his influential distinction between dementia praecox and
mania. Dementia praecox developed into the schizophrenia concept, whereas the
manias formed the basis for manic-depression and depressive disorder. Finally, the
distinction was incorporated into the DSM-III system, with the group of psychotic
disorders (incorporating schizophrenia), on the one hand, and broad group of
mood disorders (incorporating bipolar disorder and depression), on the other.
Apart from mood and psychotic disorders, three other major diagnostic
categories can be found in the DSM system: autism spectrum disorders (compris-
ing patients with self-oriented behavior and abnormal social contacts), anxiety
disorders, and the broad group of personality disorders. It is paramount for any
researcher to be aware of the fact that the DSM-III and DSM-IV systems do not
take into account any (hypothesized) cause or process which underlies the diag-
nostic categories. The DSM system defines ‘‘diagnostic categories’’ by defining
CLINICAL UTILITY OF SERUM BIOMARKERS FOR MAJOR PSYCHIATRIC DISORDERS 357
A. A DIAGNOSTIC CATEGORY
specific underlying biological reality. As a result, it is not likely that any biomarker
will reliably define a single DSM diagnostic category. This aspect is underscored
by the fact that most psychiatric research efforts result in identification of associa-
tions showing considerable overlap with the control group used. In other words,
although an association may be significant, the effect size is usually small which
leads to limits in the clinical utility. A recent example of this phenomenon was seen
in a study by Stefansson et al. (2009), which was published in Nature. This study
presents the results of a whole-genome association study of a very large group of
patients (N ¼ 12,945) with DSM-IV-defined schizophrenia compared to a control
population (N ¼ 34,591). Associations in the patient population with seven gene
variants were found [mainly related to the human leukocyte antigen (HLA)
system], with high levels of significance. However, the odds ratios of these
associations were small, ranging from 1.15 to 1.24. From a scientific point of
view, this is an important study, but it has little or no use for clinical diagnostics.
The second limitation related to the identification of biomarkers in DSM-
defined diagnostic categories is of a practical nature. If a biomarker was shown to
be specifically related to a DSM diagnostic category, then this would be a major
scientific breakthrough. However, such a finding would be trivial from a clinical
diagnostic point of view, because then the diagnostic category would already be
defined by the DSM criteria itself. This does not imply that DSM categories are
completely useless for identifying biomarkers. For example, they may serve as a
starting point for identifying subgroups within the DSM categories, as outlined in
the next subsection.
measure in these patients the biomarkers associated with the fundamental syn-
dromes, and classify patients according to their resemblance, if any, with either of
the fundamental biomarkers. The next step would be to investigate whether patients
in whom a biomarker profile sufficiently resembles one of the fundamental profiles
have a prognosis or treatment response fitting one of the fundamental syndromes.
This is, in part, the approach our group has taken when identifying a biomarker
profile for schizophrenia and by showing that a similar profile was present
in subjects who developed schizophrenia later on (Schwarz et al., 2010, 2011).
The previous two subsections dealt with the subject of diagnosis of psychiatric
illnesses. Biomarkers may also be used for prediction of treatment response or the
risk associated with psychiatric drug treatment or, conversely, to indicate a low
likelihood of spontaneous improvement (Laughren, 2010). The main goal of
biomarker application in predicting efficacy and risk is to subgroup the population
into responders and nonresponders and into subgroups containing those who are
at risk or not at risk for some adverse event of interest. Examples of possible
biomarkers include imaging measures, serum assays, genetic assays (genomic
markers), physiological measures, histopathological findings, psychological tests,
and demographic variables (age, gender, race).
There are two principal ways a biomarker could be used to subdivide the
population, such as on the basis of differences in exposure or differences in
pharmacodynamic response. In either case, the differences could divide patients
on the basis of either efficacy or risk (Laughren, 2010). For example, if biomarker-
positive patients differ from biomarker-negative patients by having higher expo-
sures to a drug, this difference could translate into a difference in efficacy
(e.g., better efficacy in biomarker-positive patients) or a difference in risk (e.g., a
greater risk in biomarker-positive patients). Similarly, a pharmacodynamic differ-
ence between biomarker-positive and -negative patients, unrelated to exposure,
could be reflected by differences in efficacy or risk.
There are already some examples of genomic biomarkers that predict exposure
such as pharmacokinetic differences based on the different activities in metabolizing
enzymes. These enzymes include CYP2C9, CYP2B6, CYP2C19, and CYP2D6
(Laughren, 2010). Atomoxetine, a selective norepinephrine reuptake inhibitor
approved for the treatment of attention deficit hyperactivity disorder (ADHD), is
cleared predominantly by CYP2D6, and subjects who are poor at metabolizing
2D6 have 10-fold higher plasma levels of atomoxetine compared to those who are
good 2D6 metabolizers (Sauer et al., 2005). It is also known that 2D6 poor
metabolizers have approximately eightfold increases in plasma levels of desipramine
after exposure to this drug, compared to 2D6 high metabolizers (Brosen et al., 1986).
CLINICAL UTILITY OF SERUM BIOMARKERS FOR MAJOR PSYCHIATRIC DISORDERS 361
Table I
PROBLEM BASINS ACCORDING TO VAN PRAAG (2000).
Syndromes characterized by disturbed reality testing and clear consciousness (a basin containing,
among others, the group of schizophrenia psychoses)
Syndromes characterized by disturbed reality testing and lowered consciousness (including the group
of so-called organic psychoses)
Syndromes characterized by disturbances in affect regulation among which the emphasis might be on
mood, anxiety, or aggression dysregulation
Syndromes characterized by disturbed cognition, among which information storage and/or
information retrieval and/or information appraisal may be the main seat of impairment
Conditions in which social adaptation and affiliative abilities are disturbed; a basin containing the
various personality disorders
Conditions with disturbed impulse regulation, comprising, among others, the eating disorders and
certain disorders in aggression regulation
Syndromes characterized by ‘‘termination pathology’’—that is, inability to terminate behaviors at an
appropriate point in time—a group holding, among others, obsessive-compulsive and the addiction
disorders
Somatic syndromes without manifest somatic pathology—a basin that would house, among others, the
somatiform and sexual disorders
CLINICAL UTILITY OF SERUM BIOMARKERS FOR MAJOR PSYCHIATRIC DISORDERS 363
Recent years have seen an increase in interest in the notion that psychiatric
disorders do not come in stable ‘‘cross-sectional’’ entities but that they are
dynamic in nature and not unlike the stages that can be identified in the develop-
ment of malignancies. Conceptualizing schizophrenia as a neurodevelopmental
disorder implies the notion of trajectory of illness. Figure 1 (Insel, 2010) shows this
trajectory and the supposed underlying biological alterations.
Based on the neurodevelopmental model of schizophrenia, four stages of
schizophrenia can be hypothesized from risk to prodrome, psychosis, and chronic
disability (McGorry et al., 2008). At present, diagnosis is based on the symptoms
and signs of psychosis. The advent of biomarkers and new cognitive tools as well
as the identification of subtle clinical features may enable the detection of earlier
stages of risk and prodrome (Nestler and Hyman, 2010) and the identification of
specific illness trajectories.
The earliest stage is risk, before detectable deficits occur. McGorry and his
colleagues (Henry et al., 2010) have established that the prodrome of schizophrenia is
a valid second stage of the illness before psychosis. The prodrome is identified based
on changes in thoughts (e.g., bizarre ideas falling short of psychotic ideation), social
isolation, and impaired functioning (e.g., reduced performance at school). Some of
these features seem endemic to adolescence and the problem of distinguishing a
high risk for psychosis from more common adolescent angst remains a challenge.
Although a structured interview was developed to aid in the identification of high
risk for psychosis (Woods et al., 2009), the addition of biomarkers may enhance
detection and increase the predictive power. Given the high rate of behavioral
distress in adolescence and the likelihood that many with prodromal symptoms
will either grow out of these or develop other disorders, the challenge is to increase
sensitivity for detecting ultrahigh risk while not sacrificing specificity. Specificity is a
challenge as many of those who seek help for prodromal symptoms will actually
develop other forms of psychopathology and not schizophrenia.
In the oncology field, a key study was published by Valk et al. (2004), entitled
‘‘Prognostically useful gene-expression profiles in acute myeloid leukemia.’’ The
issues present in oncology, as well as the methodology used, might be relevant for
364 NICO J.M. VAN BEVEREN AND WITTE J.G. HOOGENDIJK
60
Arborization
40
Myelination
20 Prefrontal
inhibitory synapses
0
Fertilization 0 5 10 15 20 25
Age (years)
Stage IV:
chronic
B Stage I: risk Stage II: prodome Stage III: psychosis disability
<12 years 12–18 years 18–24 years >24 years
100
Prefrontal Deficient
Percentage of maximum
20
Prefrontal
inhibitory synapses
0
5 10 15 20 25
Age (years)
FIG. 1. Neurodevelopmental model of schizophrenia (Insel, 2010; reprinted with permission). (A) Nor-
mal cortical development involves proliferation, migration, arborization (circuit formation), and myelina-
tion. The first two processes occur mostly during prenatal life, and the latter two continue through the first
two decades of life. The combined effects of pruning of the neuronal arbor and myelin deposition are
thought to account for the progressive reduction of gray matter observed with longitudinal neuroimaging.
(B) The trajectory in children developing schizophrenia could include reduced elaboration of inhibitory
pathways and excessive pruning of excitatory pathways leading to an altered excitatory–inhibitory
balance in the prefrontal cortex. Reduced myelination would alter connectivity. Although some data
support each of these possible neurodevelopmental mechanisms for schizophrenia, none has been proven
to cause the syndrome. Detection of prodromal neurodevelopmental changes could permit early inter-
vention with potential prevention or preemption of psychosis.
‘‘Acute myeloid leukemia is not a single disease but a group of neoplasms with
diverse genetic abnormalities and variable responses to treatment.’’ The authors
addressed an important issue in the field, namely, that the methods used at the
time for (sub)classifying AML had limited value for prognosis and guiding treat-
ment selection. It was known that the outcome of AML could in part be predicted
by assessing the cytological characteristics of the tumor cells. However, these
traditionally used characteristics correlated only partially with outcome. Although
it was recognized that these different outcome trajectories were probably depen-
dent on differential underlying molecular characteristics, the mechanisms were
poorly recognized. Thus, this situation shows some relationship with psychiatric
disorders if we substitute ‘‘major psychiatric syndrome’’ for AML, and ‘‘pheno-
typical criteria as used in DSM-IV’’ for cytological characteristics.
The Valk et al. (2004) paper addressed this issue by taking the gene-expression
patterns of the tumor cells as a starting point, without an a priori hypothesis about
the specific alterations. Using a group of 285 patients, the gene-expression
patterns were clustered, based on their overall similarity. This allowed the identi-
fication of subclusters of patients with similar gene-expression patterns. A total of
16 subgroups were thus identified. These subgroups redefined the previous sub-
classifications and showed better predictive value with respect to prognosis.
This approach resembles the ‘‘broad-to-narrow’’ method that we described
earlier and exemplifies the utility of unsupervised ‘‘bottom-up’’ clustering meth-
ods in identifying novel subcategories in heterogeneous syndromes, based on
molecular profiles. Although there are clear limitations to this approach when
using serum proteins or gene-expression patterns for psychiatric disorders, some
conceptual elements may prove useful in psychiatry.
Over the past decade, converging results from postmortem research, neuroim-
aging, genetic association studies, and measurements of peripheral blood status
have pointed to the presence of several ‘‘biological themes’’ within the broad
context of the schizophrenia syndrome and, to a lesser extent, within the bipolar
disorder syndrome. These themes can be summarized as ‘‘altered glucose metab-
olism’’ (Ryan et al., 2003; Spelman et al., 2007; Venkatasubramanian et al., 2007;
Van Nimwegen et al., 2008; Fernandez-Egea et al., 2008, 2009; Guest et al., 2010),
‘‘immunological alterations’’ (for a review, see Drexhage et al., 2010), ‘‘altered
presence of growth factors’’ (for a review, see Van Beveren et al., 2006),
366 NICO J.M. VAN BEVEREN AND WITTE J.G. HOOGENDIJK
and depressive episodes (Cunha et al., 2006; de Oliveira et al., 2009). As seen in
schizophrenia, BDNF levels appear to return to normal in successfully treated
individuals (Tramontina et al., 2009).
In major depression, there are long-standing observations of alterations in
HPA axis function (Holsboer and Barden, 1996; Nemeroff and Vale, 2005).
Evidence for hyperactivity of the HPA-axis was shown by higher daytime cortisol
levels, nonsuppression after dexamethasone ingestion and higher corticotropin-
releasing hormone, and adrenocorticotropin hormone levels among persons with
depression (Carroll et al., 1976; Gold et al., 1995; Pfohl et al., 1985; Stokes et al.,
1984), and recently confirmed by Vreeburg et al. (2009). However, inconsistencies
are present in these studies, which most likely results from the heterogeneity of the
depression syndrome. Higher cortisol levels have been reported frequently in
studies of medicated inpatients with severe melancholic or psychotic depression.
Also in major depression, convincing evidence for a (pro)inflammatory status has
also been observed which includes increased levels of IL-1 and IL-6 (Catena-
Dell’Osso et al., 2011). The relationship between inflammation and depression has
been underscored by the prominence of depressive symptoms following the acute
and chronic administration of cytokines such as interferon-alpha. Altered levels of
cytokines have also been described in bipolar disorder, although to a lesser extent
as seen in schizophrenia. Elevated levels of TNF have been described, as well as
some evidence for increased levels of IL-1 and IL-6 (O’Brien et al., 2006; Brietzke
et al., 2009; Kunz et al., 2011).
Therefore, there are robust findings showing alterations in peripheral mole-
cules in major mental disorders. However, the measurement of single molecules
has limited value as clinically useful biomarkers due to the substantial degree of
overlap between patients and control, which is typically seen, resulting in limited
sensitivity and specificity. Nevertheless, there are important proof-of-principle
aspects related to these findings. The first of these is that alterations in several
peripheral molecules are indeed present and with sufficient statistical effect sizes.
The second of these is that alterations of some molecules covary with the severity
of symptom dimensions (i.e., BDNF with the presence of positive psychotic
symptoms), suggesting a causative relationship and emphasizing the possible
utility of peripheral markers to identify symptoms dimensions. The final aspect
is that the altered molecules reflect several of the biological themes that have been
previously identified in genetic, postmortem, and neuroimaging research on the
etiology of major psychiatric disorders, suggesting that subclustering of major
psychiatric disorders based on these biological themes may be feasible.
To achieve this, multiple molecules should be investigated simultaneously, in a
high-throughput manner, enabling functional subclustering within the patient
groups. Thus far, a limited number of publications have described such a multi-
analyte approach.
368 NICO J.M. VAN BEVEREN AND WITTE J.G. HOOGENDIJK
A more comprehensive, and possibly more fruitful, approach has been facili-
tated by a relatively recent development, namely, the investigation of multiple
serum/plasma molecules at the same time. This is the approach of using multi-
protein arrays or mass-spectroscopy profiling platforms (see chapters, ‘‘Proteomic
technologies for biomarker studies in psychiatry: advances and needs’’ by Mar-
tins-de-Souza et al. and ‘‘The application of multiplexed assay systems for molec-
ular diagnostics’’ by Schwarz et al.). It is expected that the results of these
approaches will allow for a more reliable separation of patients with a specific
diagnosis from controls and from other diagnostic groups compared to the use of
single molecule biomarkers. Additionally, it opens up the possibility for the
nonhypothesis driven subclustering of patients originating from broad diagnostic
categories, such as the approach described in the section above (Section IV).
Multi-analyte and array profiling techniques enable the simultaneous detec-
tion of hundreds of proteins with high sensitivity and accuracy and can be
successfully applied to identify biomarkers (or clusters of markers) that correlate
with disease (see Ray et al., 2007 for an application in Alzheimer’s disease). At
present, a limited number of studies have taken this approach. Domenici et al.
(2010) have shown the results of a study investigating 245 patients with major
depression, 229 patients with schizophrenia, and 254 controls, using a platform
that allows for the simultaneous detection of 79 molecules including several
cytokines, neurotrophins, and metabolic proteins. They reported identification
of a cluster of molecules which gave a good degree of specificity for major
depression versus controls, and ‘‘superior discriminative power’’ for schizophrenia
versus controls. However, a major limitation of this study was that patients were
medicated and subgroup analysis was not performed.
Schwarz et al. (2010, 2011) used a similar but extended platform of molecular
assays, allowing for the simultaneous measurement of 181 molecules. In a more
elaborate approach, involving a majority of never-medicated schizophrenia
patients, they measured the levels of these molecules in serum from 250 first
and recent onset schizophrenia patients, as well as patients with major depressive
disorder, euthymic bipolar disorder, Asperger syndrome, and 280 control subjects.
These analyses resulted in identification of a signature comprising 34 analytes
allowing for the separation of over 80% of the schizophrenia subjects from
controls across five independent cohorts. The authors concluded that a biological
signature for schizophrenia can be identified in serum (Schwarz et al., 2011). This
study laid the groundwork for development of the first commercially available test
(VeriPsychTM, see chapter ‘‘Algorithm development for diagnostic biomarker
assays’’ by Izmailov et al.) aiding in the diagnosis of schizophrenia, and for
distinguishing schizophrenia patients from healthy controls and from those
CLINICAL UTILITY OF SERUM BIOMARKERS FOR MAJOR PSYCHIATRIC DISORDERS 369
Acknowledgments
This chapter was written while the authors were employed by the Erasmus MC
Medical Center Rotterdam, The Netherlands. There was no other funding
source. Many thanks to the members of the Bahn group (especially Sabine
Bahn and Emanuel Schwarz) and to Lieuwe de Haan, Wim van den Brink, and
Aart-jan Beekman for their interesting insights on issues of clinical diagnostics.
Thanks to Peter Sillevis Smitt for his data on the relationship between anti-
NMDA receptor encephalitis and psychiatric symptoms.
370 NICO J.M. VAN BEVEREN AND WITTE J.G. HOOGENDIJK
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THE FUTURE: BIOMARKERS, BIOSENSORS, NEUROINFORMATICS,
AND E-NEUROPSYCHIATRY
Christopher R. Lowe
Department of Chemical Engineering and Biotechnology, Institute of Biotechnology,
University of Cambridge, Cambridge, United Kingdom
Abstract
I. Introduction
II. Current Diagnostic Tools for Mental Illness
III. The Emergence of Molecular Biomarkers
IV. Clinical Impact of Biomarkers
V. Point-of-Care Testing
VI. Biosensors
VII. Biosensors in Neuroscience
VIII. Neuroinformatics
IX. e-Neuroscience and e-Neuropsychiatry
Acknowledgments
References
Abstract
I. Introduction
primarily due to the fact that the fundamental lesions underlying diseases of the
central nervous system (CNS) are ill established and likely to be accentuated by
complex perturbations and dysregulation of regulatory mechanisms, protein
expression profiles, and metabolic pathways. Consequently, any understanding
of these disorders should be accompanied with a systems level view of the
dysregulated factors contributing to the pathogenesis. It is imperative to construct
disease- or symptom-specific molecular fingerprints or panels of molecular bio-
markers by integrating multiple ‘‘omics’’ databases established from genomics,
proteomics, and metabolomics studies on disease versus control samples (see
chapters ‘‘The utility of gene expression in blood cells for diagnosing neuropsy-
chiatric disorders’’ by Woelk et al. and ‘‘Proteomic technologies for biomarker
studies in psychiatry: advances and needs’’ by Martins-de-Souza et al.).
A molecular biomarker is a chemical, physical, or biological parameter that
can be objectively measured and evaluated quantitatively as an indicator of
normal or pathogenic states, to measure the onset or progress of disease, of
compliance or response to a therapeutic intervention (Quinones and Kaddurah-
Daouk, 2009). Biomarkers can be specific cells, genes, gene products, such as
mRNA transcripts, proteins, and their posttranslational modifications, enzymes,
hormones, peptides, or metabolites (Craig-Shapiro et al., 2008).
The discovery of authenticated biomarkers for psychological, neuropsychia-
tric, and neurodegenerative disorders could dramatically change the future deliv-
ery of mental health care if they are incorporated into standard operating systems
and clinical decision making. Appropriate genomic, proteomic, and metabolomic
biomarkers should allow precise prediction of disease susceptibility and risk,
diagnosis and prognosis, patient and therapeutic stratification, patient response,
and adverse drug reactions, and ensure compliance (Fig. 1).
Genes abnormally regulated during neuropsychiatric disorders or affected by
drug treatments may help to define aberrant cellular processes and serve as
diagnostics and novel druggable targets. Several recent studies using cDNA
microarray and differential display techniques have investigated changes in gene
expression in AD (Hata et al., 2001; Pasinetti, 2001), seizures and epilepsy
(Aronica et al., 2001; French et al., 2001), PD (Chun et al., 2001), schizophrenia
(Mirnics et al., 2000), bipolar disorder (Sun et al., 2001; Le-Niculescu et al., 2007),
and other neurological disorders (Kontkanen and Castrén, 2002). However,
despite progress on functional genomics for neuropsychiatric disorders, significant
challenges remain before expression profiling can form a reliable basis for specific
and sensitive biomarkers. Neuropsychiatric diseases are complex polygenic dis-
orders with variable penetrance, whose phenotypic heterogeneity, overlap, and
interdependence are not well understood and which are greatly influenced by
epigenetic modifications, stress, lifestyle, infections, and medications (Danziger
et al., 2005; Crow, 2007 (Fig. 2).
380 CHRISTOPHER R. LOWE
Compliance testing
Disease evolution
Prognosis Patient
stratification
FIG. 1. Implication of biomarkers and biosensors in the disease evolution of psychological, neu-
ropsychiatric, and neurodegenerative disorders.
Genome
CNS
Behaviour Environment
CSF Proteome
Metabolome
Blood
Biomarkers
FIG. 2. Schematic of the interactions between genetic and epigenetic factors and behavior and their
effect on the discovery of genomic, proteomic, and metabolomic biomarkers from brain biopsies,
cerebrospinal fluid (CSF), and blood.
V. Point-of-Care Testing
A typical centralized hospital laboratory has evolved over the past 100 years
into a fully automated system of bar-coded patient identification, sample collec-
tion, sample pretreatment, and passage through high-throughput multiplexed
clinical chemistry and immunoassay platforms, with results becoming available
minutes, hours, or days later. Such delays can hamper timely diagnosis, impede
decision making, and affect outcomes (see chapter ‘‘Challenges of introducing
new biomarker products for neuropsychiatric disorders into the market’’ by Bahn
et al.). However, in recent years, there has been a quiet revolution in diagnostics
practice, with a perceptible trend in taking tests to the patient, rather than the
patient to the tests. Substantial technological advances in assay chemistry, sensor
and transducer configurations, electronic processing and data manipulation,
384 CHRISTOPHER R. LOWE
Pre-late
twentieth century
Physician’s
office
Home Primary
Clinical Healthcare care
diagnostics provision centre
Regional
hospital
Post-late Community
twentieth century care
Local centre
hospital
FIG. 3. Evolution of health-care provision from the home in the late twentieth century to the
regional hospital via various community and local services in the late twentieth century and anticipated
full circle back to the home in the early twenty-first century. Clinical diagnostics are following a similar
pattern of development.
BIOMARKERS, BIOSENSORS, NEUROINFORMATICS, AND E-NEUROPSYCHIATRY 385
devices and the larger table- or bench-top instruments. The majority of handheld
systems comprise a disposable reagent prefilled strip incorporated into a cartridge
or cassette to facilitate addition of the sample, whence following development of
the test, the resulting signal is interpreted visually or measured using an inexpen-
sive reader or meter. Current POC tests cover critical care (blood gases, ions),
emergency medicine (troponin), risk assessment (cholesterol), diabetes manage-
ment (glucose, hemoglobin A1c), personal well-being (pregnancy, fertility), lifestyle
(alcohol, drugs of abuse), and infectious diseases (human immunodeficiency virus,
sexually transmitted infections) in a market estimated to be worth $18.7B by 2013
(Kricka and Thorpe, 2010). Such tests usually comprise a reagent-impregnated
paper strip or pad, lateral flow devices, dipsticks, cards, slides, flow-through tubes,
cartridges, and cassettes with inbuilt meters and displays. A good example of state-
of-the-art consumer electronics technology for POC testing is the introduction of
the Clearblue Digital Pregnancy testÒ by SPD, which provides both an unambig-
uous readout of the result (pregnant/not pregnant) and an indication of the
number of weeks since conception.
VI. Biosensors
A key element for the development of POC systems is the biosensor. Biosensor
technology is an analytical platform which can empower physicians or psychia-
trists with the ability to confirm a suspected diagnosis on the spot when the patient
presents him or herself and thereby obviate the requirement to attend hospital.
A biosensor is an analytical device which combines a biorecognition system with a
suitable physicochemical transducer to convert the recognition of the target
analyte directly into an electrical signal (Lowe et al., 1992; Gizeli and Lowe,
1996; Lowe, 2007a,b). Figure 4 shows a schematic biosensor comprising (i) a
biorecognition system, such as a natural or an engineered enzyme, antibody,
receptor, nucleic acid, aptamer, peptide, or other synthetic biomimetic, microor-
ganism, organelle, or tissue, in close conjunction with (ii) a transducer, which
transforms the signal resulting from the interaction of the analyte with the
biorecognition system into an electrical system, and (iii) the associated electronics
and signal processing to display the results in a user-friendly fashion. The trans-
ducer can exploit any physical principle based on electrochemical, optical, acous-
tic, magnetic, thermal, or microegineered devices (Lowe, 1999, 2007a,b).
Classic examples of biosensors include amperometric glucose sensors for
diabetes management (Foulds and Lowe, 1988; Wolowacz et al., 1992), conducti-
metric devices (Cullen et al., 1990), surface plasmon resonance (SPR; Cullen et al.,
1987/1988), the resonant mirror (Buckle et al., 1993; Davies et al., 1994;
386 CHRISTOPHER R. LOWE
Analyte
Electrical
(Bio)recognition system
Optical
Transducer Acoustic
Magnetic
Thermal
Microelectronic
Instrumentation
signal processing
output Microengineered
FIG. 4. Schematic of a generalized biosensor showing the (bio)recognition system identifying the
analyte in a complex biological sample, the physic-chemical transducer for conversion of the recogni-
tion into an electrical signal, and the instrumentation for outputting the signal in the desired
user-friendly fashion.
Watts et al., 1994), fiber optic sensors (Carlyon et al., 1992; Tubb et al., 1995, 1997;
Hale et al., 1996; Schipper et al., 1997), Mach-Zehnder interferometry (Schipper
et al., 1997), planar waveguides (Mayr et al., 2009), and various optical grating
(Erdélyi et al., 2007) and acoustic and microcantilever devices (Gizeli et al., 1992;
Stevenson and Lowe, 1999; Sindi et al., 2001; Stevenson et al., 2001, 2003, 2006;
Haefliger and Boisen, 2007). More recent trends in biosensor technology include
the use of aptamers (Brody and Gold, 2000; Strehlitz et al., 2008; Abe et al., 2011),
peptides (Huang and Koide, 2010), molecularly imprinted polymers (Haupt and
Belmont, 2007), and genetically engineered binding proteins (Ge et al., 2003) and
enzymes (Campas et al., 2009) as more durable, selective, and higher affinity
recognition elements, the use of electropolymerization to immobilize biomole-
cules in thin films on sensor surfaces (Cosnier, 2007), metal (Elghanian et al., 1997)
and magnetic nanoparticles (Yellen and Erb, 2007), quantum dots (Abramowitz,
2007) to amplify signals, and conducting polymer nanowire- (Wanekaya et al.,
2007) and carbon nanotube-based sensors (Barone et al., 2007) to aid in miniatur-
ization of sensor formats.
Perhaps the largest single advance in biosensor technology for clinical and
POC analysis involves microfluidics and lab-on-a-chip platforms (Li, 2010). This
technology was first demonstrated with a fully integrated GC and thermal
BIOMARKERS, BIOSENSORS, NEUROINFORMATICS, AND E-NEUROPSYCHIATRY 387
conductivity detector on a 4-in. silicon wafer in 1979 (Terry et al., 1979), while the
first liquid separation was exemplified in 1989 (Sethi et al., 1989) and a high-
performance liquid chromatography (HPLC) and conductimetric detector was
fabricated on Si-Pyrex in 1990 (Manz et al., 1990). A miniaturized chemical
analysis platform based on capillary electrophoresis (CE) linked to laser-induced
fluorescence (LIF) detection was fabricated on a glass chip with channels 10 mm
deep and 30 mm wide and used to resolve biomolecules in 6 min (Manz et al.,
1992). Since then, a plethora of different clinical, cellular, protein, and nucleic
acid analytes have been resolved and quantified using these techniques in devices
fabricated in silicon, glass, plastics, and polydimethylsiloxane using on-chip CE,
dielectrophoresis, optical trapping, filters, pumps, and valves (Li, 2010; Napoli
et al., 2010). For example, DNA extraction; amplification by thermal, isothermal,
and rolling circle polymerase chain reaction (PCR); and subsequent hybridiza-
tion, sequencing, and genotyping have been facilitated by the use of a microfluidic
system implemented on a plastic monolithic platform (Liu et al., 2002). Similarly,
microfluidic devices have been actively developed for clinical diagnostics, protein
separation and functional assay, proteomics and immunoassay (Li, 2010). Thus,
homogeneous (Chen et al., 2002), competitive (Schmalzing et al., 1997), and
heterogeneous (Ko et al., 2003) immunoassays and multiplexed label-free assays
(Carlborg et al., 2010) have been developed.
The long cherished ambition to witness biosensors displacing all other analyt-
ical technologies within the health-care and biomedical sectors has yet to be
realized after nearly three decades of intensive research and the introduction of
a vast armamentarium of new devices and techniques. The most attractive
features of the biosensor, that is, its small size, ruggedness, inexpensiveness, low
power burden, rapid and real-time response, use by lay personnel, biocompatibili-
ty, and ready interface with computer and mobile technology has yet to be
translated into reality. However, biosensors are beginning to emerge as a solution
to resolving challenging issues in fundamental neuroscience and as a means to
reduce the complexity of multiplexed genomic and proteomic biomarker assays
and bring the diagnosis of multigenic neurological diseases closer to the patient
(Bell and Kornguth, 2007).
Chemical signaling plays a key role in neural function although few investi-
gators directly measure the concentrations of neurotransmitters and modulators
in the extracellular space. Biosensors offer the prospect of measuring neurotrans-
mitter production with adequate temporal (milliseconds) and spatial (100 nm–
10 mm) resolution (Dale et al., 2005). Key targets such as pH, pO2, glutamate,
388 CHRISTOPHER R. LOWE
The most popular platform for the detection and quantitation of neurodegen-
erative biomarkers uses the phenomenon of SPR (Cullen et al., 1987/1988). For
example, a nanoscale optical sensor based on SPR has been exploited to quanti-
tate the interaction between the amyloid-b-derived diffusible ligands (ADDLs)
and specific anti-ADDL antibodies (Haes et al., 2005), while the BiacoreÒ SPR
system has been used to characterize the polyglutamine tracts as threshold
biomarkers for the onset of AD (Bennett et al., 2002). Similarly, fiber optic and
SPR array technologies have been suggested as ways to multiplex multiple assays
on the same biological sample (Gašparac and Walt, 2007; Suzuki et al., 2007).
More recently, a flexible new grating-coupled SPR sensor system that combines
the joint advantages of discretely functionalized, encoded microparticles and
label-free detection has been reported (Kastl et al., 2008). This system offers the
prospect of simultaneously investigating the real-time binding kinetics of a variety
of molecular interactions in a single multiplexed platform; thus, one multiplexed
assay could employ a wide range of immobilization chemistries, surface prepara-
tion methods, and formats. The new system offers a very high level of assay
conformability to the end user, particularly when compared to fixed microarrays
and permits the contemporaneous assay of both genomic and proteomic
biomarkers (Kastl et al., 2010).
The third, and most challenging option, for the diagnosis of psychological,
neuropsychiatric, and neurodegenerative disorders, is to reduce the complexity
and cost of these multiparametric assay panels to permit POC testing in the hands
of frontline physician’s and psychiatrists. There are a variety of simplified tests
being developed for field use, that is, outside the controlled laboratory environ-
ment. One example of such a test is the development of simple reflection holo-
grams that combine an analyte-selective ‘‘smart’’ polymer with optical
interrogation by eye or instrumentation and a reporting diffraction grating trans-
ducer (Blyth et al., 1996). They are fabricated by passing a single collimated laser
beam through a silver halide emulsion coated onto a glass or plastic substrate
backed with a silvered mirror. Interference between the ingoing and outgoing
beams creates a standing wave interference pattern, which after development and
fixing, forms a series of fringes of silver grains 20 nm in diameter separated by a
distance equal to 1/2l of the laser light used in their construction and distributed
within the thickness of the emulsion ( 5–10 mm). Under white light illumination,
the fringes act like a Bragg diffraction grating and reflect a narrow band of
wavelength governed by Bragg’s law:
ml ¼ 2n@cosy
where m is the diffraction order, l is the wavelength of light in vacuo, n is the
average refractive index of the system, @ is the spacing between the fringes, and y
is the angle between the incident light and the diffracting planes. Any physical,
chemical, or biological interaction which alters the fringe spacing (@) by swelling
390 CHRISTOPHER R. LOWE
or contraction, the average refractive index (n) or the total number or distribution
of the fringes within the thickness of the film will result in observable changes in
the wavelength (color) or intensity (brightness) of the hologram. It has been
demonstrated that through the rational design of the hydrogel, which incorpo-
rates suitable receptors, that volumetric changes can be induced on binding the
complementary target analyte. Thus, responsive holograms for gases, ions, meta-
bolites, inhibitors, drugs, enzymes, antigens, antibodies, and whole cells have been
created based on this principle (Mayes et al., 1999, 2002; Marshall et al., 2003,
2004; Lee et al., 2004; Sartain et al., 2006; Lowe, 2007a,b; Tan and Lowe, 2009;
Martinez-Hurtado et al., 2010). Responsive holograms of this type are ideally
suited to POC applications since they are designed react to specific stimuli; are
inexpensive to manufacture by mass producible techniques; are miniaturizable;
generate a readout in real time; can be configured as planar, fiber optic, or
particulate arrays; and display low or no power burden (Martinez-Hurtado
et al., 2010). It is anticipated that these devices will be configured as a handheld
card for use by the physician or psychiatrist to aid in the diagnosis of psychologi-
cal, neuropsychiatric, and neurodegenerative disorders.
Microfluidic devices also offer the prospect of inexpensive POC tests suitable
for execution by lay personnel with small sample sizes (Whitesides, 2006). Such
devices have been fabricated on thin and flexible films (Focke et al., 2010) and have
used manual torque-operated valves for sandwich immunoassays (Weibel et al.,
2005) and thread as a versatile material for low-cost microfluidic diagnostics
(Li et al., 2010; Reches et al., 2010).
VIII. Neuroinformatics
The final part of the jigsaw relates to mobile communication and the internet
which will allow unstructured data to be organized, interpreted, evaluated, and
reformatted for facile presentation to the end user, be they the physician, psychia-
trist, or patient. Frontline analysis, that is, near or on-patient collection of data via
real-time or multiplexed sensor arrays, coupled with artificial intelligence and
mobile communication systems could bring diagnosis, application, and internet-
based services back to the patient (Fig. 5; Ryhänen et al., 2010). The prospect of
ambient intelligence, in which sensing, computation, and communication are
universally available and ready to serve the end user in an intelligent way, is
predicated on intelligent mobile devices embedded in all aspects of the human
environment, home, office, and public places. The concept of remote health care
is particularly relevant to psychological, neurodegenerative, and neuropsychiatric
disorders, since these are long-term debilitating conditions which require constant
monitoring and treatment and afflict many of the more developed regions of the
world. Telepsychiatry is already an established approach where the patient is in
his own home or office, particularly in rural or underserved regions, and can
access the psychiatrist via Webcam or high-speed internet. Similarly, there have
been developments in emergency e-psychiatry to provide consultation for suicidal,
homicidal, violent, psychotic, depressed, manic, and acutely anxious psychiatric
patients (Shore et al., 2007). Such emergency e-psychiatry services can be provided
to hospital A&E departments, jails, community mental health centers, substance
abuse clinics, and schools.
392 CHRISTOPHER R. LOWE
Physician’s office
Emergency room
bedside
Data flow
Neuroinformatic flow Substance abuse
centre
JAIL
Data store/
server
Patient
Drug automat
PRESCRIPTION
MEDICINES
OTHER HEALTHCARE SERVICES
FIG. 5. e-Neuroscience: A future scenario in which psychiatric and neurodegenerative patients are
monitored with selective wireless biosensors and the data is transmitted to the psychiatrist for
interpretation and onward passage to key service providers and drug automats prior to release of
prescription medicines and other health-care services to the patient.
Acknowledgments
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Subject Index
401
402 SUBJECT INDEX
Schizophrenia. See also Immune and Serotonin and noradrenaline reuptake inhibitor
neuroimmune alterations, schizophrenia (SNRI), 338
and mood disorder (cont.) Serum biomarkers, major psychiatric disorders
cortisol levels, 157 categories, diagnostic
HPA axis dysfunction, 156 identification, 359
leptin receptor, 157 overlap, control group, 358–359
metabolic perturbations, 156 clinical examples
spectrum disorders, 156 adolescent, adjustment disorder, 353
psychotic symptoms, 96 another young female, schizophrenia,
in silico functional pathway analysis, 99–101 354–355
special considerations, 160 young female, schizophrenia, 353–354
syndromal diagnostic procedures, 96–97 description, 352
therapeutic implications diagnosis
adipocyte differentiation, 159 categories, DSM, 356–357, 358, 361
Alzheimer’s disease, 159 DSM-III system, 356
antipsychotic drugs, 158–159 ‘‘first-rank symptoms’’, 359–360
DHEA, 160 molecules identified, peripheral blood, 352
hyperinsulinemia, 158–159 multiplex molecular measurements
insulin-sensitizing agents, 159 multi-analyte and array profiling
metabolic dysfunction, 159 techniques, 368
positive and negative symptoms, schizophrenia patients, 368–369
158–159 serum/plasma molecules, 368
PPAR-gamma signaling, 159 oncology
type-1 and type-2 immune system AML, 364–365
CNS, evidence, 130–131 gene-expression patterns, tumor cells, 365
innate immune response, 127–128 peripheral blood-based molecules, 369
Schizophrenia (SZ) problem basins, Van Praag, 362–363
attenuated flush response, 260 single molecular measurements
caspase 3 activity, 246–247 alterations, peripheral molecules, 367
dopamine D2 receptor antagonists, 263 BDNF, 366–367
dysregulated pathways, 246–247 ‘‘biological themes’’, 365–366
insulin levels, 271 HPA axis function, 367
PANSS, 265 schizophrenia patients, 366–367
proinsulin molecules, 272 specific developmental trajectories
serum signature, 260–261 schizophrenia neurodevelopmental model,
ventricular volume, 243–244 363, 364
SELDI-TOF schizophrenia prodrome, 363
MALDI, 79 specific treatment response
proteome analyses, CSF, 79 application goal, 360
Selective reaction monitoring (SRM) HER2 gene, 361
experiments, 83–84 metabolizing enzymes, 360
MRM, 83–84 population subdivision, 360
nature and multiplexing capability, 84 SSRI, 361
proteomic studies, 84 use
steps, 83–84 anti-NMDA receptor encephalitis, 355–356
Selective serotonin reuptake inhibitors (SSRIs) bipolar disorder, 355
citalopram, 361 schizophrenia development, 355
G/G allele, 339 Shotgun proteomics
polymorphic serotonin transporter gene description, 74–75
allele, 361 in vivo labeling (see In vivo labeling)
SUBJECT INDEX 411
413
414 CONTENTS OF RECENT VOLUMES
The Role of MST Neurons during Ocular Brain Development and Generation of Brain
Tracking in 3D Space Pathologies
K. Kawano, U. Inoue, A. Takemura, Gregory L. Holmes and Bridget McCabe
Y. Kodaka, and F. A. Miles Maturation of Channels and Receptors: Conse-
Visual Navigation in Flying Insects quences for Excitability
M. V. Srinivasan and S.-W. Zhang David F. Owens and Arnold R. Kriegstein
Neuronal Matched Filters for Optic Flow Neuronal Activity and the Establishment of
Processing in Flying Insects Normal and Epileptic Circuits during Brain
H. G. Krapp Development
A Common Frame of Reference for the Analysis John W. Swann, Karen L. Smith, and Chong L. Lee
of Optic Flow and Vestibular Information The Effects of Seizures of the Hippocampus of
B. J. Frost and D. R. W. Wylie the Immature Brain
Optic Flow and the Visual Guidance of Ellen F. Sperber and Solomon L. Moshe
Locomotion in the Cat Abnormal Development and Catastrophic
H. Sherk and G. A. Fowler Epilepsies: The Clinical Picture and Relation to
Stages of Self-Motion Processing in Primate Neuroimaging
Posterior Parietal Cortex Harry T. Chugani and Diane C. Chugani
F. Bremmer, J.-R. Duhamel, S. B. Hamed, and Cortical Reorganization and Seizure Generation
W. Graf in Dysplastic Cortex
G. Avanzini, R. Preafico, S. Franceschetti,
Optic Flow Analysis for Self-Movement
Perception G. Sancini, G. Battaglia, and V. Scaioli
C. J. Duffy Rasmussen’s Syndrome with Particular Refer-
Neural Mechanisms for Self-Motion Perception ence to Cerebral Plasticity: A Tribute to Frank
in Area MST Morrell
Fredrick Andermann and Yuonne Hart
R. A. Andersen, K. V. Shenoy, J. A. Crowell,
and D. C. Bradley Structural Reorganization of Hippocampal
Computational Mechanisms for Optic Flow Networks Caused by Seizure Activity
Analysis in Primate Cortex Daniel H. Lowenstein
M. Lappe Epilepsy-Associated Plasticity in gamma-
Human Cortical Areas Underlying the Percep- Amniobutyric Acid Receptor Expression,
Function and Inhibitory Synaptic Properties
tion of Optic Flow: Brain Imaging Studies
M. W. Greenlee Douglas A. Coulter
What Neurological Patients Tell Us about the Synaptic Plasticity and Secondary Epilepto-
Use of Optic Flow genesis
Timothy J. Teyler, Steven L. Morgan,
L. M. Vaina and S. K. Rushton
Rebecca N. Russell, and Brian L. Woodside
INDEX
Synaptic Plasticity in Epileptogenesis: Cel-
lular Mechanisms Underlying Long-Lasting
Synaptic Modifications that Require New Gene
Expression
Volume 45
Oswald Steward, Christopher S. Wallace, and
Paul F. Worley
Mechanisms of Brain Plasticity: From Normal
Cellular Correlates of Behavior
Brain Function to Pathology
Emma R. Wood, Paul A. Dudchenko, and
Philip. A. Schwartzkroin
Howard Eichenbaum
418 CONTENTS OF RECENT VOLUMES
Clinical Implications of Circulating Neuroster- Processing Human Brain Tissue for in Situ
oids Hybridization with Radiolabelled Oligonucleo-
Andrea R. Genazzani, Patrizia Monteleone, tides
Massimo Stomati, Francesca Bernardi, Louise F. B. Nicholson
Luigi Cobellis, Elena Casarosa, Michele Luisi,
In Situ Hybridization of Astrocytes and Neurons
Stefano Luisi, and Felice Petraglia
Cultured in Vitro
Neuroactive Steroids and Central Nervous L. A. Arizza-McNaughton, C. De Felipe,
System Disorders and S. P. Hunt
Mingde Wang, Torbjörn Bäckström,
In Situ Hybridization on Organotypic Slice
Inger Sundström, Göran Wahlström,
Cultures
Tommy Olsson, Di Zhu, Inga-Maj Johansson,
A. Gerfin-Moser and H. Monyer
Inger Björn, and Marie Bixo
Quantitative Analysis of in Situ Hybridization
Neuroactive Steroids in Neuropsychopharma-
Histochemistry
cology
Andrew L. Gundlach and Ross D. O’Shea
Rainer Rupprecht and Florian Holsboer
Current Perspectives on the Role of Neuroster- Part II: Nonradioactive in Situ hybridization
oids in PMS and Depression Nonradioactive in Situ Hybridization Using Alka-
Lisa D. Griffin, Susan C. Conrad, and line Phosphatase-Labelled Oligonucleotides
Synthia H. Mellon S. J. Augood, E. M. McGowan, B. R. Finsen,
INDEX B. Heppelmann, and P. C. Emson
Combining Nonradioactive in Situ Hybridization
with Immunohistological and Anatomical
Techniques
Volume 47
Petra Wahle
Nonradioactive in Situ Hybridization: Simplified
Introduction: Studying Gene Expression in
Procedures for Use in Whole Mounts of Mouse
Neural Tissues by in Situ Hybridization
and Chick Embryos
W. Wisden and B. J. Morris
Linda Ariza-McNaughton and Robb Krumlauf
Part I: In Situ Hybridization with Radiolabelled
INDEX
Oligonucleotides
In Situ Hybridization with Oligonucleotide
Probes
Wl. Wisden and B. J. Morris
Cryostat Sectioning of Brains Volume 48
Victoria Revilla and Alison Jones
Processing Rodent Embryonic and Early Post- Assembly and Intracellular Trafficking of
natal Tissue for in Situ Hybridization with GABAA Receptors Eugene
Radiolabelled Oligonucleotides Barnes
David J. Laurie, Petra C. U. Schrotz,
Subcellular Localization and Regulation of
Hannah Monyer, and Ulla Amtmann GABAA Receptors and Associated Proteins
Processing of Retinal Tissue for in Situ Hybrid- Bernhard Lüscher and Jean-Marc Fritschy D1
ization Dopamine Receptors
Frank Müller Richard Mailman
Processing the Spinal Cord for in Situ Hybridiza- Molecular Modeling of Ligand-Gated Ion
tion with Radiolabelled Oligonucleotides Channels: Progress and Challenges
A. Berthele and T. R. Tölle Ed Bertaccini and James R. Trudel
420 CONTENTS OF RECENT VOLUMES
Alzheimer’s Disease: Its Diagnosis and Patho- The Treatment of Infantile Spasms: An
genesis Evidence-Based Approach
Jillian J. Kril and Glenda M. Halliday Mark Mackay, Shelly Weiss, and
DNA Arrays and Functional Genomics in O. Carter Snead III
Neurobiology ACTH Treatment of Infantile Spasms: Mechan-
Christelle Thibault, Long Wang, Li Zhang, and isms of Its Effects in Modulation of Neuronal
Michael F. Miles Excitability
K. L. Brunson, S. Avishai-Eliner, and
INDEX
T. Z. Baram
Neurosteroids and Infantile Spasms: The Deox-
Volume 49 ycorticosterone Hypothesis
Michael A. Rogawski and Doodipala S. Reddy
What Must We Know to Develop Better Infantile Spasms: Criteria for an Animal Model
Therapies? Carl E. Stafstrom and Gregory L. Holmes
Jean Aicardi INDEX
CONTENTS OF RECENT VOLUMES 421
Polyol Pathway and Diabetic Peripheral Neu- Glucose, Stress, and Hippocampal Neuronal
ropathy Vulnerability
Peter J. Oates Lawrence P. Reagan
422 CONTENTS OF RECENT VOLUMES
Volume 52
Volume 53
Neuroimmune Relationships in Perspective
Frank Hucklebridge and Angela Clow Section I: Mitochondrial Structure and Function
Sympathetic Nervous System Interaction with Mitochondrial DNA Structure and Function
the Immune System Carlos T. Moraes, Sarika Srivastava,
Virginia M. Sanders and Adam P. Kohm Ilias Kirkinezos, Jose Oca-Cossio,
Mechanisms by Which Cytokines Signal the Brain Corina van Waveren, Markus Woischnick,
Adrian J. Dunn and Francisca Diaz
Section III: Secondary Respiratory Chain The Mitochondrial Theory of Aging: Involve-
Disorders ment of Mitochondrial DNA Damage and
Friedreich’s Ataxia Repair
J. M. Cooper and J. L. Bradley Nadja C. de Souza-Pinto and
Vilhelm A. Bohr
Wilson Disease
INDEX
C. A. Davie and A. H. V. Schapira
Hereditary Spastic Paraplegia
Christopher J. McDermott and Pamela J. Shaw
Volume 54
Cytochrome c Oxidase Deficiency
Giacomo P. Comi, Sandra Strazzer,
Sara Galbiati, and Nereo Bresolin Unique General Anesthetic Binding Sites Within
Distinct Conformational States of the Nicotinic
Section IV: Toxin Induced Mitochondrial Acetylcholine Receptor
Dysfunction Hugo R. Ariaas, William, R. Kem,
James R. Truddell, and Michael P. Blanton
Toxin-Induced Mitochondrial Dysfunction
Susan E. Browne and M. Flint Beal Signaling Molecules and Receptor Transduction
Cascades That Regulate NMDA Receptor-
Section V: Neurodegenerative Disorders Mediated Synaptic Transmission
Suhas. A. Kotecha and John F. MacDonald
Parkinson’s Disease
L. V. P. Korlipara and A. H. V. Schapira Behavioral Measures of Alcohol Self-Administra-
tion and Intake Control: Rodent Models
Huntington’s Disease: The Mystery Unfolds?
Herman H. Samson and Cristine L. Czachowski
Åsa Petersén and Patrik Brundin
Dopaminergic Mouse Mutants: Investigating
Mitochondria in Alzheimer’s Disease
the Roles of the Different Dopamine Receptor
Russell H. Swerdlow and Stephen J. Kish
Subtypes and the Dopamine Transporter
Contributions of Mitochondrial Alterations, Shirlee Tan, Bettina Hermann, and
Resulting from Bad Genes and a Hostile Envi- Emiliana Borrelli
ronment, to the Pathogenesis of Alzheimer’s
Drosophila melanogaster, A Genetic Model System
Disease
for Alcohol Research
Mark P. Mattson
Douglas J. Guarnieri and Ulrike Heberlein
Mitochondria and Amyotrophic Lateral
INDEX
Sclerosis
Richard W. Orrell and Anthony H. V. Schapira
Proteomic Approaches in Drug Discovery and Neuroimaging Studies in Bipolar Children and
Development Adolescents
Holly D. Soares, Stephen A. Williams, Rene L. Olvera, David C. Glahn, Sheila C. Caetano,
Peter J. Snyder, Feng Gao, Tom Stiger, Steven R. Pliszka, and Jair C. Soares
Christian Rohlff, Athula Herath, Trey Sunderland, Chemosensory G-Protein-Coupled Receptor
Karen Putnam, and W. Frost White Signaling in the Brain
Section III: Informatics Geoffrey E. Woodard
INDEX
Volume 66
Neural Modeling and Functional Brain Imaging: Georg Winterer, Ahmad R. Hariri, David Goldman,
The Interplay Between the Data-Fitting and and Daniel R. Weinberger
Simulation Approaches
Neuroreceptor Imaging in Psychiatry: Theory
Barry Horwitz and Michael F. Glabus
and Applications
Combined EEG and fMRI Studies of Human W. Gordon Frankle, Mark Slifstein, Peter S. Talbot,
Brain Function and Marc Laruelle
V. Menon and S. Crottaz-Herbette
INDEX
INDEX
Volume 68
Volume 67
Fetal Magnetoencephalography: Viewing the
Distinguishing Neural Substrates of Heterogen- Developing Brain In Utero
eity Among Anxiety Disorders Hubert Preissl, Curtis L. Lowery, and Hari Eswaran
Jack B. Nitschke and Wendy Heller Magnetoencephalography in Studies of Infants
Neuroimaging in Dementia and Children
K. P. Ebmeier, C. Donaghey, and Minna Huotilainen
N. J. Dougall Let’s Talk Together: Memory Traces Revealed
Prefrontal and Anterior Cingulate Contributions by Cooperative Activation in the Cerebral
to Volition in Depression Cortex
Jack B. Nitschke and Kristen L. Mackiewicz Jochen Kaiser, Susanne Leiberg, and Werner
Lutzenberger
Functional Imaging Research in Schizophrenia
H. Tost, G. Ende, M. Ruf, F. A. Henn, and Human Communication Investigated With
A. Meyer-Lindenberg Magnetoencephalography: Speech, Music, and
Gestures
Neuroimaging in Functional Somatic Syn-
Thomas R. Knösche, Burkhard Maess, Akinori
dromes
Nakamura, and Angela D. Friederici
Patrick B. Wood
Combining Magnetoencephalography and
Neuroimaging in Multiple Sclerosis
Functional Magnetic Resonance Imaging
Alireza Minagar, Eduardo Gonzalez-Toledo,
Klaus Mathiak and Andreas J. Fallgatter
James Pinkston, and Stephen L. Jaffe
Beamformer Analysis of MEG Data
Stroke
Arjan Hillebrand and Gareth R. Barnes
Roger E. Kelley and Eduardo Gonzalez-Toledo
Functional Connectivity Analysis in Magnetoen-
Functional MRI in Pediatric Neurobehavioral
cephalography
Disorders
Alfons Schnitzler and Joachim Gross
Michael Seyffert and F. Xavier Castellanos
Human Visual Processing as Revealed by Mag-
Structural MRI and Brain Development
netoencephalographys
Paul M. Thompson, Elizabeth R. Sowell,
Yoshiki Kaneoke, Shoko Watanabe, and Ryusuke
Nitin Gogtay, Jay N. Giedd, Christine N. Vidal,
Kakigi
Kiralee M. Hayashi, Alex Leow, Rob Nicolson,
Judith L. Rapoport, and Arthur W. Toga A Review of Clinical Applications of Magne-
toencephalography
Neuroimaging and Human Genetics
CONTENTS OF RECENT VOLUMES 429
Andrew C. Papanicolaou, Eduardo M. Castillo, Eric D. Young, Jane J. Yu, and Lina A. J. Reiss
Rebecca Billingsley-Marshall, Ekaterina Pataraia,
Spectral Processing in the Inferior Colliculus
and Panagiotis G. Simos
Kevin A. Davis
INDEX
Neural Mechanisms for Spectral Analysis in the
Auditory Midbrain, Thalamus, and Cortex
Monty A. Escabı́ and Heather L. Read
Volume 69
Spectral Processing in the Auditory Cortex
Mitchell L. Sutter
Nematode Neurons: Anatomy and Anatomical
Methods in Caenorhabditis elegans Processing of Dynamic Spectral Properties of
David H. Hall, Robyn Lints, and Zeynep Altun Sounds
Adrian Rees and Manuel S. Malmierca
Investigations of Learning and Memory in
Caenorhabditis elegans Representations of Spectral Coding in the
Andrew C. Giles, Jacqueline K. Rose, and Catharine Human Brain
H. Rankin Deborah A. Hall, PhD
Neural Specification and Differentiation Spectral Processing and Sound Source Deter-
Eric Aamodt and Stephanie Aamodt mination
Donal G. Sinex
Sexual Behavior of the Caenorhabditis elegans
Male Spectral Information in Sound Localization
Scott W. Emmons Simon Carlile, Russell Martin, and Ken McAnally
Volume 70 Volume 71
Spectral Processing by the Peripheral Auditory Autism: Neuropathology, Alterations of the GA-
System Facts and Models BAergic System, and Animal Models
Enrique A. Lopez-Poveda Christoph Schmitz, Imke A. J. van Kooten,
Patrick R. Hof, Herman van Engeland,
Basic Psychophysics of Human Spectral Pro- Paul H. Patterson, and Harry W. M. Steinbusch
cessing
Brian C. J. Moore The Role of GABA in the Early Neuronal
Development
Across-Channel Spectral Processing Marta Jelitai and Emı´lia Madarasz
John H. Grose, Joseph W. Hall III, and
Emily Buss GABAergic Signaling in the Developing Cere-
bellum
Speech and Music Have Different Requirements Chitoshi Takayama
for Spectral Resolution
Robert V. Shannon Insights into GABA Functions in the Developing
Cerebellum
Non-Linearities and the Representation of Audi- Mo´nica L. Fiszman
tory Spectra
430 CONTENTS OF RECENT VOLUMES
ECT and the Youth: Catatonia in Context Understanding Myelination through Studying its
Frank K. M. Zaw Evolution
Rüdiger Schweigreiter, Betty I. Roots,
Catatonia in Autistic Spectrum Disorders: A
Medical Treatment Algorithm Christine Bandtlow, and Robert M. Gould
Max Fink, Michael A. Taylor, and Neera Ghaziuddin INDEX
Psychological Approaches to Chronic
Catatonia-Like Deterioration in Autism
Spectrum Disorders Volume 74
Amitta Shah and Lorna Wing
Section V: Blueprints Evolutionary Neurobiology and Art
Blueprints for the Assessment, Treatment, and C. U. M. Smith
Future Study of Catatonia in Autism Spectrum Section I: Visual Aspects
Disorders
Perceptual Portraits
Dirk Marcel, Dhossche, Amitta Shah, and Lorna Wing
Nicholas Wade
INDEX
The Neuropsychology of Visual Art: Conferring
Capacity
Volume 73 Anjan Chatterjee
Vision, Illusions, and Reality
Chromosome 22 Deletion Syndrome and Christopher Kennard
Schizophrenia Localization in the Visual Brain
Nigel M. Williams, Michael C. O’Donovan, and George K. York
Michael J. Owen
Section II: Episodic Disorders
Characterization of Proteome of Human Cere-
Neurology, Synaesthesia, and Painting
brospinal Fluid Amy Ione
Jing Xu, Jinzhi Chen, Elaine R. Peskind,
Jinghua Jin, Jimmy Eng, Catherine Pan, Fainting in Classical Art
Thomas J. Montine, David R. Goodlett, and Philip Smith
Jing Zhang Migraine Art in the Internet: A Study of 450
Hormonal Pathways Regulating Intermale and Contemporary Artists
Interfemale Aggression Klaus Podoll
Neal G. Simon, Qianxing Mo, Shan Hu, Sarah Raphael’s Migraine with Aura as Inspir-
Carrie Garippa, and Shi-Fang Lu ation for the Foray of Her Work into Abstraction
Neuronal GAP Junctions: Expression, Function, Klaus Podoll and Debbie Ayles
and Implications for Behavior The Visual Art of Contemporary Artists with
Clinton B. McCracken and David C. S. Roberts Epilepsy
Effects of Genes and Stress on the Neurobiology Steven C. Schachter
of Depression Section III: Brain Damage
J. John Mann and Dianne Currier
Creativity in Painting and Style in Brain-
Quantitative Imaging with the Micropet Small- Damaged Artists
Animal Pet Tomograph Julien Bogousslavsky
Paul Vaska, Daniel J. Rubins, David L. Alexoff, and
Wynne K. Schiffer Artistic Changes in Alzheimer’s Disease
432 CONTENTS OF RECENT VOLUMES
Sebastian J. Crutch and Martin N. Rossor Karen Beckett and Mary K. Baylies
Section IV: Cerebrovascular Disease Organization of the Efferent System and Struc-
Stroke in Painters ture of Neuromuscular Junctions in Drosophila
H. Bäzner and M. Hennerici Andreas Prokop
Amy J. Jak, Katherine J. Bangen, Christina E. GluK1 Receptor Antagonists and Hippocampal
Wierenga, Lisa Delano-Wood, Jody Corey-Bloom, Mossy Fiber Function
and Mark W. Bondi Robert Nisticò, Sheila Dargan, Stephen M. Fitzjohn,
Proton Magnetic Resonance Spectroscopy in David Lodge, David E. Jane, Graham L. Collingridge,
Dementias and Mild Cognitive Impairment and Zuner A. Bortolotto
H. Randall Griffith, Christopher C. Stewart, and Monoamine Transporter as a Target Molecule
Jan A. den Hollander for Psychostimulants
Ichiro Sora, BingJin Li, Setsu Fumushima, Asami Fukui,
Application of PET Imaging to Diagnosis
Yosefu Arime, Yoshiyuki Kasahara, Hiroaki Tomita, and
of Alzheimer’s Disease and Mild Cognitive
Kazutaka Ikeda
Impairment
James M. Noble and Nikolaos Scarmeas Targeted Lipidomics as a Tool to Investigate
Endocannabinoid Function
The Molecular and Cellular Pathogenesis Giuseppe Astarita, Jennifer Geaga, Faizy Ahmed, and
of Dementia of the Alzheimer’s Type: An Daniele Piomelli
Overview
Francisco A. Luque and Stephen L. Jaffe The Endocannabinoid System as a Target for
Novel Anxiolytic and Antidepressant Drugs
Alzheimer’s Disease Genetics: Current Status Silvana Gaetani, Pasqua Dipasquale, Adele Romano,
and Future Perspectives Laura Righetti, Tommaso Cassano, Daniele Piomelli,
Lars Bertram and Vincenzo Cuomo
Frontotemporal Lobar Degeneration: Insights GABAA Receptor Function and Gene
from Neuropsychology and Neuroimaging Expression During Pregnancy and Postpartum
Andrea C. Bozoki and Muhammad U. Farooq Giovanni Biggio, Maria Cristina Mostallino, Paolo
Follesa, Alessandra Concas, and Enrico Sanna
Lewy Body Dementia
Early Postnatal Stress and Neural Circuit
Jennifer C. Hanson and Carol F. Lippa
Underlying Emotional Regulation
Dementia in Parkinson’s Disease Machiko Matsumoto, Mitsuhiro Yoshioka,
Bradley J. Robottom and William J. Weiner and Hiroko Togashi
Current Techniques and Concepts in Peripheral Gene Therapy Perspectives for Nerve Repair
Nerve Repair Serena Zacchigna and Mauro Giacca
Maria Siemionow and Grzegorz Brzezicki
Use of Stem Cells for Improving Nerve
Artificial Scaffolds for Peripheral Nerve Regeneration
Reconstruction Giorgio Terenghi, Mikael Wiberg, and
Valeria Chiono, Chiara Tonda-Turo, and Paul J. Kingham
Gianluca Ciardelli Transplantation of Olfactory Ensheathing Cells
Conduit Luminal Additives for Peripheral for Peripheral Nerve Regeneration
Nerve Repair Christine Radtke, Jeffery D. Kocsis, and Peter M. Vogt
Hede Yan, Feng Zhang, Michael B. Chen, and
Manual Stimulation of Target Muscles has
William C. Lineaweaver
Different Impact on Functional Recovery after
Tissue Engineering of Peripheral Nerves Injury of Pure Motor or Mixed Nerves
Bruno Battiston, Stefania Raimondo, Pierluigi Tos, Nektarios Sinis, Thodora Manoli, Frank Werdin,
Valentina Gaidano, Chiara Audisio, Anna Scevola, Armin Kraus, Hans E. Schaller, Orlando
Isabelle Perroteau, and Stefano Geuna Guntinas-Lichius, Maria Grosheva, Andrey Irintchev,
Mechanisms Underlying The End-to-Side Nerve Emanouil Skouras, Sarah Dunlop, and
Regeneration Doychin N. Angelov
Eleana Bontioti and Lars B. Dahlin Electrical Stimulation for Improving Nerve
Regeneration: Where do we Stand?
Experimental Results in End-To-Side
Tessa Gordon, Olewale A. R. Sulaiman, and
Neurorrhaphy
Adil Ladak
Alexandros E. Beris and Marios G. Lykissas
Phototherapy in Peripheral Nerve Injury:
End-to-Side Nerve Regeneration: From the Effects on Muscle Preservation and Nerve
Laboratory Bench to Clinical Applications Regeneration
Pierluigi Tos, Stefano Artiaco, Igor Papalia, Shimon Rochkind, Stefano Geuna, and
Ignazio Marcoccio, Stefano Geuna, and Asher Shainberg
Bruno Battiston
Age-Related Differences in the Reinnervation
after Peripheral Nerve Injury
Uroš Kovačič, Janez Sketelj, and Fajko F. Bajrović
444 CONTENTS OF RECENT VOLUMES
Behavioral Outcome Measures for the Assess- Transcranial Sonography in the Premotor Diag-
ment of Sensorimotor Function in Animal nosis of Parkinson’s Disease
Models of Movement Disorders Stefanie Behnke, Ute Schroder and Daniela Berg
Sheila M. Fleming
Pathophysiology of Transcranial Sonography
The Role of DNA Methylation in the Central Signal Changes in the Human Substantia Nigra
Nervous System and Neuropsychiatric Disorders K. L. Double, G. Todd and S. R. Duma
Jian Feng and Guoping Fan
Transcranial Sonography for the Discrimination
Heritability of Structural Brain Traits: An of Idiopathic Parkinson’s Disease from the Atyp-
Endo-phenotype Approach to Deconstruct ical Parkinsonian Syndromes
Schizophrenia A. E. P. Bouwmans, A. M. M. Vlaar, K. Srulijes,
Nil Kaymaz and J. Van Os W. H. Mess AND W. E. J. Weber
The Role of Striatal NMDA Receptors in Drug Transcranial Sonography in the Discrimination
Addiction of Parkinson’s Disease Versus Vascular Parkin-
Yao-Ying Ma, Carlos Cepeda, and Cai-Lian Cui sonism
Pablo Venegas-Francke
Deciphering Rett Syndrome With Mouse Gen-
etics, Epigenomics, and Human Neurons TCS in Monogenic Forms of Parkinson’s Disease
Jifang Tao, Hao Wu, and Yi Eve Sun Kathrin Brockmann and Johann Hagenah
Part III—Transcranial Sonography in other
INDEX
Movement Disorders and Depression
Transcranial Sonography in Brain Disorders
Volume 90 with Trace Metal Accumulation
Uwe Walter
Part I: Introduction Transcranial Sonography in Dystonia
Introductory Remarks on the History and Cur- Alexandra Gaenslen
rent Applications of TCS Transcranial Sonography in Essential Tremor
Matthew B. Stern Heike Stockner and Isabel Wurster
Method and Validity of Transcranial Sonogra- VII—Transcranial Sonography in Restless Legs
phy in Movement Disorders Syndrome
David Školoudı́k and Uwe Walter Jana Godau and Martin Sojer
Transcranial Sonography—Anatomy Transcranial Sonography in Ataxia
Heiko Huber Christos Krogias, Thomas Postert and Jens Eyding
Part II: Transcranial Sonography in Parkinsons Transcranial Sonography in Huntington’s Disease
Disease Christos Krogias, Jens Eyding and Thomas Postert
Transcranial Sonography in Relation to SPECT Transcranial Sonography in Depression
and MIBG Milija D. Mijajlovic
Yoshinori Kajimoto, Hideto Miwa and Tomoyoshi
Kondo Part IV: Future Applications and Conclusion
Diagnosis of Parkinson’s Disease—Transcranial Transcranial Sonography-Assisted Stereotaxy
Sonography in Relation to MRI and Follow-Up of Deep Brain Implants in
Ludwig Niehaus and Kai Boelmans Patients with Movement Disorders
Uwe Walter
Early Diagnosis of Parkinson’s Disease
Alexandra Gaenslen and Daniela Berg Conclusions
446 CONTENTS OF RECENT VOLUMES
INDEX INDEX
Volume 91
Volume 92
The Role of microRNAs in Drug Addiction: A
Big Lesson from Tiny Molecules The Development of the Science of Dreaming
Andrzej Zbigniew Pietrzykowski Claude Gottesmann
The Genetics of Behavioral Alcohol Responses Dreaming as Inspiration: Evidence from Reli-
in Drosophila gion, Philosophy, Literature, and Film
Aylin R. Rodan and Adrian Rothenfluh Kelly Bulkeley
Neural Plasticity, Human Genetics, and Risk for Developmental Perspective: Dreaming Across
Alcohol Dependence the Lifespan and What This Tells Us
Shirley Y. Hill Melissa M. Burnham and Christian Conte
Using Expression Genetics to Study the Neuro- REM and NREM Sleep Mentation
biology of Ethanol and Alcoholism Patrick Mcnamara, Patricia Johnson, Deirdre
Sean P. Farris, Aaron R. Wolen McLaren, Erica Harris,Catherine Beauharnais and
and Michael F. Miles Sanford Auerbach
Genetic Variation and Brain Gene Expression in Neuroimaging of Dreaming: State of the Art and
Rodent Models of Alcoholism: Implications for Limitations
Medication Development Caroline Kussé, Vincenzo Muto, Laura Mascetti, Luca
Karl Björk, Anita C. Hansson Matarazzo, Ariane Foret, Anahita Shaffii-Le Bourdiec
and Wolfgang H. Sommer and Pierre Maquet
Identifying Quantitative Trait Loci (QTLs) and Memory Consolidation, The Diurnal Rhythm of
Genes (QTGs) for Alcohol-Related Phenotypes Cortisol, and The Nature of Dreams: A New
in Mice Hypothesis
Lauren C. Milner and Kari J. Buck Jessica D. Payne
Glutamate Plasticity in the Drunken Amygdala: Characteristics and Contents of Dreams
The Making of an Anxious Synapse Michael Schredl
Brian A. Mccool, Daniel T. Christian,
Marvin R. Diaz and Anna K. Läck Trait and Neurobiological Correlates of Individual
Differences in Dream Recall and Dream Content
Ethanol Action on Dopaminergic Neurons in the Mark Blagrove and Edward F. Pace-Schott
Ventral Tegmental Area: Interaction with Intrin-
sic Ion Channels and Neurotransmitter Inputs Consciousness in Dreams
Hitoshi Morikawa and Richard A. Morrisett David Kahn and Tzivia Gover
Alcohol and the Prefrontal Cortex The Underlying Emotion and the Dream: Relat-
Kenneth Abernathy, L. Judson Chandler ing Dream Imagery to the Dreamer’s Under-
and John J. Woodward lying Emotion can Help Elucidate the Nature
of Dreaming
BK Channel and Alcohol, A Complicated Affair Ernest Hartmann
Gilles Erwan Martin
Dreaming, Handedness, and Sleep Architecture:
A Review of Synaptic Plasticity at Purkinje Interhemispheric Mechanisms
Neurons with a Focus on Ethanol-Induced
Stephen D. Christman and Ruth E. Propper
Cerebellar Dysfunction
C. Fernando Valenzuela, Britta Lindquist
CONTENTS OF RECENT VOLUMES 447
The Balance Between Excitation And Inhibition Huntington’s Disease: Clinical Presentation and
Treatment
And Functional Sensory Processing in the Soma-
tosensory Cortex M.J.U. Novak and S.J. Tabrizi
Zhi Zhang and Qian-Quan Sun Genetics and Neuropathology of Huntington’s
Disease: Huntington’s Disease
INDEX Anton Reiner, Ioannis Dragatsis and Paula Dietrich
Volume 98 Pathogenic Mechanisms in Huntington’s Disease
Lesley Jones and Alis Hughes
An Introduction to Dyskinesia—the Clinical
Spectrum Experimental Models of HD And Reflection on
Ainhi Ha and Joseph Jankovic Therapeutic Strategies
Olivia L. Bordiuk, Jinho Kim and Robert J. Ferrante
L-dopa-induced Dyskinesia—Clinical Presenta-
Cell-based Treatments for Huntington’s Disease
tion, Genetics, And Treatment
L.K. Prashanth, Susan Fox and Wassilios G. Meissner Stephen B. Dunnett and Anne E. Rosser
Clinical Phenomenology of Dystonia
Experimental Models of L-DOPA-induced
Dyskinesia Carlo Colosimo and Alfredo Berardelli
Tom H. Johnston and Emma L. Lane Genetics and Pharmacological Treatment of
Molecular Mechanisms of L-DOPA-induced Dystonia
Dyskinesia Susan Bressman and Matthew James
Gilberto Fisone and Erwan Bezard Experimental Models of Dystonia
New Approaches to Therapy A. Tassone, G. Sciamanna, P. Bonsi, G. Martella and
Jonathan Brotchie and Peter Jenner A. Pisani
Surgical Treatment of Dystonia
Surgical Approach to L-DOPA-induced
John Yianni, Alexander L. Green and
Dyskinesias
Tipu Z. Aziz
Tejas Sankar and Andres M. Lozano
INDEX
Clinical and Experimental Experiences of
Graft-induced Dyskinesia
Emma L. Lane
Volume 99
Tardive Dyskinesia: Clinical Presentation and
Treatment Seizure and Epilepsy: Studies of Seizure-
P.N. van Harten and D.E. Tenback disorders in Drosophila
Louise Parker, Iris C. Howlett, Zeid M. Rusan and
Epidemiology and Risk Factors for (Tardive) Mark A. Tanouye
Dyskinesia
D.E. Tenback and P.N. van Harten
450 CONTENTS OF RECENT VOLUMES
Homeostatic Control of Neural Activity: A Dros- Kinetic Behavior and Reversible Inhibition of
ophila Model for Drug Tolerance and Depend- Monoamine Oxidases—Enzymes that Many
ence Want Dead
Alfredo Ghezzi and Nigel S. Atkinson Keith F. Tipton, Gavin P. Davey and
Andrew G. McDonald
Attention in Drosophila
Bruno van Swinderen The Pharmacology of Selegiline
Kálmán Magyar
The roles of Fruitless and Doublesex in the Control
of Male Courtship Type A Monoamine Oxidase Regulates Life and
Brigitte Dauwalder Death of Neurons in Neurodegeneration and
Neuroprotection
Circadian Plasticity: from Structure to Behavior Makoto Naoi, Wakako Maruyama,
Lia Frenkel and Marı́a Fernanda Ceriani Keiko Inaba-Hasegawa and Yukihiro Akao
Learning and Memory in Drosophila: Behavior, Multimodal Drugs and their Future for
Genetics, and Neural Systems Alzheimer’s and Parkinson’s Disease
Lily Kahsai and Troy Zars Cornelis J. Van der Schyf and Werner J. Geldenhuys
Studying Sensorimotor Processing with Physiology Neuroprotective Profile of the Multitarget Drug
in Behaving Drosophila Rasagiline in Parkinson’s Disease
Johannes D. Seelig and Vivek Jayaraman Orly Weinreb, Tamar Amit, Peter Riederer,
Moussa B.H. Youdim and Silvia A. Mandel
Modeling Human Trinucleotide Repeat Diseases
in Drosophila Rasagiline in Parkinson’s Disease
Zhenming Yu and Nancy M. Bonini L.M. Chahine and M.B. Stern